Glycerol and acetic acid converting yeast cells with improved acetic acid conversion

ABSTRACT

Cell that is genetically modified comprising:
         a) one or more nucleotide sequence encoding a NAD + -dependent acetylating acetaldehyde dehydrogenase (E.C. 1.2.1.10);   b) one or more nucleotide sequence encoding a acetyl-CoA synthetase (E.C. 6.2.1.1);   c) one or more nucleotide sequence encoding a glycerol dehydrogenase (E.C. 1.1.1.6); and   d) one or more nucleotide sequence encoding a dihydroxyacetone kinase (E.C. 2.7.1.28 or E.C. 2.7.1.29).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/914,612, filed 25 Feb. 2016, which is a § 371 National StageApplication of PCT/EP2014/068324, filed 29 Aug. 2014, which claimspriority to European Patent Application No. 13182222.3, filed 29 Aug.2013. The disclosures of the priority applications are incorporated intheir entirety herein by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 CFR § § 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “Sequence_Listing_2919208-373001_ST25.txt” createdon 10 Jul. 2019, and 137,666 bytes in size) is submitted concurrentlywith the instant application, and the entire contents of the SequenceListing are incorporated herein by reference.

BACKGROUND Field of the Invention

The present invention relates to metabolic engineering in microorganismssuch as yeast. In particular the invention relates glycerol and aceticacid converting yeast cells with improved acetic acid conversion. Theinvention further relates to the processes wherein the yeast cellsproduce fermentation product such as ethanol.

Description of Related Art

Second generation bioethanol is produced from e.g. lignocellulosicfractions of plant biomass that is hydrolyzed into free monomericsugars, such as hexoses and pentoses, for fermentation into ethanol.Apart from the sugar release during pretreatment and hydrolysis of thebiomass, some toxic by-products are formed. For instance, furfural andHMF are two of these products. The quantities in which they are formeddepend on several pretreatment parameters, such as temperature, pressureand pretreatment time.

Lignocellulosic hydrolysates also contain high amounts of acetic acid,which is a potent inhibitor of the fermentative capacity ofmicroorganisms, such as yeasts.

Glycerol is the major by-product during fermentation of sugars intoethanol, mainly formed as a result of re-oxidation reactions to consumethe excess NADH formed during biosynthesis under anaerobic conditions(van Dijken and Scheffers, 1986). As a result, during industrialfermentations, about 5 to 10% of the consumed sugars by yeast cells arediverted into glycerol. Lowering the amount of this polyol is considereda promising route to increase ethanol yield. This could be achieved byadjusting the feeding rate during the fed-batch process, or by selectingstrains that produce less glycerol.

In the literature, however, several different approaches have beenreported that could help to reduce the inhibitory effect of acetic acidon the fermentation of the sugars in hydrolysates as well as (partly)solving redox balance issues upon deletion of the genes involved inglycerol production, e.g. by genetic engineering of yeasts.

Sonderegger et al (2004) disclosed the heterologous expression ofphosphotransacetylase and acetaldehyde dehydrogenase in axylose-fermenting Saccharomyces cerevisiae strain. In combination withthe native phosphoketolase, Sonderegger et al thereby created afunctional phosphoketolase pathway that is capable of net reoxidation ofNADH generated by the heterologous expression of a xylose reductase andxylitol dehydrogenase that are used for xylose utilization in thatparticular strain.

Guadalupe et al (2009) described a Saccharomyces cerevisiae strainwherein production of the by-product glycerol is eliminated by thedisruption of the endogenous NAD-dependent glycerol 3-phosphatedehydrogenase genes (GPD1 and GPD2). Expression of the E. coli mhpFgene, encoding the acetylating NAD-dependent acetaldehyde dehydrogenase,restored the ability of the gpd1gpd2 double deletion strain to growanaerobically by supplementation of the medium with acetic acid.

Yu et al (2010) constructed Saccharomyces cerevisiae strainsmetabolically engineered for improved ethanol production from glycerolby simultaneous overexpression of glycerol dehydrogenase (encoded byGCY1), dihydroxyacetone kinase (DAK1) and the glycerol uptake protein(GUP1). In a later report by the same group (Yu et al, 2011) it isdescribed that additional overexpression of ADH1 and PDC1, encodingalcohol dehydrogenase and pyruvate decarboxylase respectively, caused anincrease in growth rate and glycerol consumption under fermentativeconditions, resulting in a slightly increased final ethanol yield.

Lee and Dasilva (2006) disclosed the yeast Saccharomyces cerevisiaeengineered to produce 1,2-propanediol from glycerol by amongst othersintroducing expression of the Escherichia coli mgs and gldA genes.

The technology described by Guadelupe et al (and also in patentapplication WO 2011/010923) provides a solution for decreasing theacetic acid content of hydrolysates during fermentation of the biomasssugars and the aforementioned acetic acid into e.g. ethanol.

Further enhancement of the ability to convert acetic acid is potentiallypossible by introducing an extra NADH-generating pathway, e.g. byadditionally (over-)expressing a glycerol consumption pathway. Uponintroduction of the aforementioned GUP1-, GCY1- and DAK1-genes (Yu etal, 2010) in a yeast strain expressing an anaerobic acetic acidconversion pathway (such as e.g. described by Medina et al, 2009),acetic acid conversion should be increased in order to maintain theredox balance, leading to further increased detoxification of thehydrolysate and higher ethanol yield. The solution of Yu et al however,does not work, since the yeast glycerol dehydrogenase (encoded by GCY1)uses NADP⁺ as a co-factor, resulting in a cofactor imbalance due toinsufficient cofactor regeneration. An alternative glyceroldehydrogenase(gldA from E. coli) was tested in combination with the acetic acidreduction pathway and indeed enhanced the conversion of acetic acidunder anaerobic growth (fermentation) conditions (patent applicationWO2013/081456).

SUMMARY

It is therefore an object of the present invention to provide for yeaststhat are capable of producing ethanol from acetic acid or acetate whileretaining their abilities of fermenting hexoses (glucose, fructose,galactose, etc) as well as pentoses like xylose, as well as processeswherein these strains are used for the production of ethanol and/orother fermentation products. An object is to provide for cells, e.g.yeast cells that are capable of producing ethanol from glycerol and/orglycerol and acetic acid while retaining their abilities of fermentinghexoses (glucose, fructose, galactose, etc) as well as pentoses likexylose. Another object is to increase the production of fermentationproduct (yield, production rate or both).

One or more of the objects are attained according to the invention thatprovides a yeast cell that is genetically modified comprising:

a) one or more nucleotide sequence encoding a heterologousNAD₊-dependent acetylating acetaldehyde dehydrogenase (E.C. 1.2.1.10);

b) one or more nucleotide sequence encoding a homologous or heterologousacetyl-CoA synthetase (E.C. 6.2.1.1);

c) one or more nucleotide sequence encoding a heterologous glyceroldehydrogenase (E.C. 1.1.1.6); and

d) one or more nucleotide sequence encoding a homologous or heterologousdihydroxyacetone kinase (E.C. 2.7.1.28 or E.C. 2.7.1.29).

In an embodiment, the cell has a deletion or disruption of one or moreendogenous nucleotide sequence encoding a glycerol 3-phosphatephosphohydrolase and/or encoding a glycerol 3-phosphate dehydrogenasegene.

One or more of the above objects are attained according to theinvention.

It is clear from the examples that according to the invention improvedfermentation product production (ethanol) may be attained.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of the enzymatic reactions involved inthe conversion of glycerol and acetic acid (acetate) into ethanol.Acetate is first converted into acetyl-CoA through the yeast enzyme Acs(Acs1 and/or Acs2, encoded by the genes ACS1 and ACS2 respectively).Acetyl-CoA is then converted into acetaldehyde through the mhpF gene, ordirectly into ethanol through the bifunctional adhE enzyme from E. coli(or similar enzymes catalyzing the same conversion). Upon introductionof the glycerol consumption pathway, converting externally addedglycerol, an extra flow of NADH is generated. Deletion of the GPD1 andGPD2 genes is optional in order to avoid the intracellular production ofglycerol and the utilization of NADH by these enzymes.

The ethanol yield per consumed sugar (glucose and/or other sugars)increases due to elimination of glycerol production, ethanol generationfrom acetate/acetic acid in the medium (and always present inlignocellulosic hydrolysates) and glycerol externally added to themedium (or hydrolysate).

FIG. 2. Schematic display of the strain construction approach. INT(integration) flanks and expression cassettes (CAS), includingselectable marker, are amplified using PCR and transferred into yeast.Recombination will take place between the connectors (designated 5, a,b, c and 3 respectively in FIG. 1) resulting in the integration of thepathway in the desired location in the yeast genome (in this case,INT1). The number of genes of interest may be extended, as described inthe examples. Unique connectors were used to facilitate recombination ofthe separate expression cassettes and integration into the genome of therecipient cell.

FIG. 3. Results of screening. The residual acetic acid concentration isplotted as function of the residual acetate concentration.

FIG. 4. The residual acetic acid concentration is plotted as function ofthe residual acetate concentration. The 150 best performing strains,based on the residual acetate and glycerol concentrations as well asethanol production from glycerol and acetate, are displayed in darkgrey.

FIG. 5. Rescreening results of newly generated transformants (R1-R8) andreference strains (RN1069, RN1189 and YD01247). Eight independenttransformants were picked per transformation. Likewise, referencestrains were inoculated in eightfold. In the upper panel, the residualacetic acid (acetate) concentration is depicted after 72 hours ofincubation. In the lower panel, the residual glycerol concentration isplotted.

FIG. 6. Results of the screening. In total, 2592 strains were screened,including reference strain RN1189. Reference strain RN1189 was included27 times. The performance of reference strain RN1189 relative to theother strains (total 2592) is depicted in the figure below. The strainsare ranked as described before, where the better performing strains areindicated by a lighter color (and are closer to the bottom-left cornerof the graph). The less well performing strains are indicated by adarker color; the change in color is gradual. The exception is that thereference strain, RN1189, is indicated in the darkest color.

FIG. 7. Results of the screening. The top five of best performingstrains are shown here: 1) represents YD01247, 2) is YD01248, 3) isYD01249, 4) is YD01250.Strain 5 was not named nor tested further.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

-   SEQ ID NO: 1 adhE Escherichia coli Bifunctional    acetaldehyde-CoA/alcohol dehydrogenase (protein);-   SEQ ID NO: 2 acdH Lactobacillus plantarum Acetaldehyde dehydrogenase    (protein);-   SEQ ID NO: 3 eutE Escherichia coli Ethanolamine utilization protein    (protein);-   SEQ ID NO: 4 Lin1129 Listeria innocua Aldehyde dehydrogenase    (protein);-   SEQ ID NO: 5 adhE Staphylococcus aureus Bifunctional    acetaldehyde-CoA/alcohol dehydrogenase (protein);-   SEQ ID NO: 6 ACS2 Saccharomyces cerevisiae Acetyl-CoA ligase    (protein);-   SEQ ID NO: 7 gldA Escherichia coli Glycerol dehydrogenase (protein);-   SEQ ID NO: 8 gldA Klebsiella pneumoniae Glycerol dehydrogenase    (protein);-   SEQ ID NO: 9 gldA Enterococcus aerogenes Glycerol dehydrogenase    (protein);-   SEQ ID NO: 10 gldA Yersinia aldovae Glycerol dehydrogenase    (protein);-   SEQ ID NO: 11 DAK1 Saccharomyces cerevisiae Dihydroxyacetone kinase    (protein);-   SEQ ID NO: 12 dhaK Klebsiella pneumoniae Dihydroxyacetone kinase    (protein);-   SEQ ID NO: 13 DAK1 Yarrowia lipolytica Dihydroxyacetone kinase    (protein);-   SEQ ID NO: 14 DAK1 Schizosaccharomyces pombe Dihydroxyacetone kinase    (protein);-   SEQ ID NO: 15 Fragment containing the TDH3-promoter;-   SEQ ID NO: 16 Fragment containing the TDH1-promoter;-   SEQ ID NO: 17 Fragment containing the PGK1-terminator;-   SEQ ID NO: 18 Fragment containing the PGK1-promoter;-   SEQ ID NO: 19 Fragment containing the PRE3-promoter;-   SEQ ID NO: 20 Fragment containing the PGI1-terminator;-   SEQ ID NO: 21 Fragment containing the ENO1-promoter;-   SEQ ID NO: 22 Fragment containing the ACT1-promoter;-   SEQ ID NO: 23 Fragment containing the CYC1-terminator;-   SEQ ID NO: 24 Fragment containing the TPI1-promoter;-   SEQ ID NO: 25 Fragment containing the ATG7-promoter;-   SEQ ID NO: 26 Fragment containing the ENO1-terminator;-   SEQ ID NO: 27 Sequence of the kanMX marker and flanking regions;-   SEQ ID NO: 28 Sequence of gene disruption cassette GPD1::hphMX;-   SEQ ID NO: 29 Sequence of gene disruption cassette GPD2::natMX;-   SEQ ID NO: 30 Forward primer 5′ INT1 fragment (INT5-f);-   SEQ ID NO: 31 Reverse primer 5′ INT1 fragment (INT5-r);-   SEQ ID NO: 32 Forward primer expression cassette 1 (con5-f);-   SEQ ID NO: 33 Reverse primer expression cassette 1 (conA-r);-   SEQ ID NO: 34 Forward primer marker (conA-f);-   SEQ ID NO: 35 Reverse primer marker (conB-r);-   SEQ ID NO: 36 Forward primer expression cassette 2 (conB-f);-   SEQ ID NO: 37 Reverse primer expression cassette 2 (conC-r);-   SEQ ID NO: 38 Forward primer expression cassette 3 (conC-f);-   SEQ ID NO: 39 Reverse primer expression cassette 3 (conD-r);-   SEQ ID NO: 40 Forward primer expression cassette 4 (conD-f);-   SEQ ID NO: 41 Reverse primer expression cassette 4 (con3-r);-   SEQ ID NO: 42 Forward primer 3′ INT1 fragment (INT3-f);-   SEQ ID NO: 43 Reverse primer 3′ INT1 fragment (INT3-r);-   SEQ ID NO: 44 Sequence of plasmid p5Abbn;-   SEQ ID NO: 45 Sequence of plasmid pBCbbn;-   SEQ ID NO: 46 Sequence of plasmid pCDbbn;-   SEQ ID NO: 47 Sequence of plasmid pD3bbn-   SEQ ID NO: 48 Sequence containing the adhE (E. coli) DNA sequence    codon-pair optimized for expression in S. cerevisiae;-   SEQ ID NO: 49 Sequence containing the acdH (L. plantarum) DNA    sequence codon-pair optimized for expression in S. cerevisiae;-   SEQ ID NO: 50 Sequence containing the eutE (E. coli) DNA sequence    codon-pair optimized for expression in S. cerevisiae;-   SEQ ID NO: 51 Sequence containing the Lin1129 (L. innocua) DNA    sequence codon-pair optimized for expression in S. cerevisiae;-   SEQ ID NO: 52 Sequence containing the adhE (S. aureus) DNA sequence    codon-pair optimized for expression in S. cerevisiae;-   SEQ ID NO: 53 Sequence containing the ACS2 (S. cerevisiae) DNA    sequence codon-pair optimized for expression in S. cerevisiae;-   SEQ ID NO: 54 Sequence containing the gldA (E. coli) DNA sequence    codon-pair optimized for expression in S. cerevisiae;-   SEQ ID NO: 55 Sequence containing the gldA (K. pneumoniae) DNA    sequence codon-pair optimized for expression in S. cerevisiae;-   SEQ ID NO: 56 Sequence containing the gldA (E. aerogenes) DNA    sequence codon-pair optimized for expression in S. cerevisiae;-   SEQ ID NO: 57 Sequence containing the gldA (Y. aldovae) DNA sequence    codon-pair optimized for expression in S. cerevisiae;-   SEQ ID NO: 58 Sequence containing the DAK1 (S. cerevisiae) DNA    sequence codon-pair optimized for expression in S. cerevisiae;-   SEQ ID NO: 59 Sequence containing the dhaK (K. pneumoniae) DNA    sequence codon-pair optimized for expression in S. cerevisiae;-   SEQ ID NO: 60 Sequence containing the DAK1 (Y. lipolytica) DNA    sequence codon-pair optimized for expression in S. cerevisiae;-   SEQ ID NO: 61 Sequence containing the DAK1 (S. pombe) DNA sequence    codon-pair optimized for expression in S. cerevisiae;

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

Saccharomyces cerevisiae produces ethanol from sugars, such as glucose,under anaerobic conditions. This process is redox-neutral. When yeast isgrowing however, a surplus of NADH is generated. In order to restore theredox balance, yeast will produce glycerol. During this process, NADH isconverted into NAD+ again. The ethanol industry considers glycerol anundesired by-product. Omission of glycerol formation under anaerobicconditions is a long felt desire from the ethanol industry. As describedabove, several attempts have been made by several different groups toredirect the carbon flux from glycerol formation towards ethanol,thereby increasing the ethanol yield.

The most direct measure to prevent glycerol formation would be deletinggenes encoding proteins involved in the biosynthesis of glycerol.However, when the GPD1 and GPD2 genes are disrupted, the yeast is unableto grow under anaerobic conditions, as it is unable to restore its redoxbalance. Medina et al (2009) demonstrated that upon introduction of aNADH-dependent acetyl-CoA dehydrogenase gene (such as the E. colimhpF-gene, as described by Medina et al), the ability of a gpd1 gpd2double deletion strain to grow under anaerobic conditions is recovered,provided that acetic acid is supplied into the fermentation medium. Theacetic acid is converted into acetyl-CoA through the ACS1/ACS2 geneproducts. Acetyl-CoA is converted into acetaldehyde, and subsequentlyinto ethanol through the mhpF and ADH1 gene products (Medina et al,2009). In this way, formation of an unwanted by-product (glycerol) iseliminated, resulting in a higher ethanol yield.

As acetic acid is often considered to be the most toxic compound presentin hydrolysates, especially in hydrolysates with a pH close to or belowthe pKa of acetic acid (pKa HAc˜4.76), there is a desire to furtherdecrease the acetate (acetic acid) concentration in hydrolysates. Oneway of increasing the anaerobic acetate conversion potential of theyeast is by introducing a glycerol conversion pathway. By introductionof a glycerol pathway that converts externally added glycerol, as thegpd1 gpd2 cell does not produce glycerol itself, even more NADH isgenerated, forcing the yeast cell to convert more acetic acid in orderto maintain the redox balance (see FIGS. 1 and WO2013/081456).

Glycerol is available in sufficiently large quantities at biorefineries.

To this end, the genes gldA from E. coli and DAK1 from S. cerevisiaewere overexpressed, in order to allow for a further increase in theconversion of the toxic acetic acid into ethanol (WO 2013/081456).Indeed, higher ethanol yields were obtained.

In order to even further improve the anaerobic (co-)conversion ofglycerol and acetic acid both in terms of rate and amount, alternativegene combinations were tested. For a number of enzymes in the pathway,i.e. glycerol dehydrogenase, dihydroxyacetone kinase and acetaldehydedehydrogenase, multiple alternative genes were tested that could furtherenhance the ability of the yeast strain to convert glycerol and aceticacid, next to pentose and hexose sugars, into ethanol under anaerobicconditions.

The invention therefore provides yeast cell that is genetically modifiedcomprising:

a) one or more nucleotide sequence encoding a NAD₊-dependent acetylatingacetaldehyde dehydrogenase (E.C. 1.2.1.10);

b) one or more nucleotide sequence encoding a acetyl-CoA synthetase(E.C. 6.2.1.1);

c) one or more nucleotide sequence encoding a glycerol dehydrogenase(E.C. 1.1.1.6); and

d) one or more nucleotide sequence encoding a dihydroxyacetone kinase(E.C. 2.7.1.28 or E.C. 2.7.1.29).

Embodiments of the invention are described below. The following itemsdescribe several embodiments of the invention, wherein amongst othersthe features a) to d) here above are detailed:

-   Item 1:-   Cell that is genetically modified comprising:    -   a) one or more nucleotide sequence encoding a NAD₊-dependent        acetylating acetaldehyde dehydrogenase (E.C. 1.2.1.10);    -   b) one or more nucleotide sequence encoding a acetyl-CoA        synthetase (E.C. 6.2.1.1);    -   c) one or more nucleotide sequence encoding a glycerol        dehydrogenase (E.C. 1.1.1.6); and    -   d) one or more nucleotide sequence encoding a dihydroxyacetone        kinase (E.C. 2.7.1.28 or E.C. 2.7.1.29).-   Item 1a:-   Cell that is genetically modified comprising:    -   a) one or more nucleotide sequence encoding a heterologous        NAD₊-dependent acetylating acetaldehyde dehydrogenase (E.C.        1.2.1.10);    -   b) one or more nucleotide sequence encoding a homologous or        heterologous acetyl-CoA synthetase (E.C. 6.2.1.1);    -   c) one or more nucleotide sequence encoding a heterologous        glycerol dehydrogenase (E.C. 1.1.1.6); and    -   d) one or more nucleotide sequence encoding a homologous or        heterologous dihydroxyacetone kinase (E.C. 2.7.1.28 or E.C.        2.7.1.29).-   Item 2.    -   Cell according to item 1, comprising a deletion or disruption of        one or more endogenous nucleotide sequence encoding a glycerol        3-phosphate phosphohydrolase (GPP1,GPP2) and/or encoding a        glycerol 3-phosphate dehydrogenase gene (GPD1, GPD2);-   Item 3.    -   Cell according to item 1 or item 2, wherein    -   b) is one or more heterologous nucleotide sequence encoding a        homologous or heterologous acetyl-CoA synthetase (E.C. 6.2.1.1)        represented by SEQ ID NO: 6 or a functional homologue of SEQ ID        NO: 6 having sequence identity of at least 60% with SEQ ID NO:        6;-   Item 4. Cell according to item 3, wherein    -   c) is one or more nucleotide sequence encoding a heterologous        glycerol dehydrogenase (E.C. 1.1.1.6) represented by SEQ ID NO:        7 or a functional homologue of SEQ ID NO: 7 having sequence        identity of at least 60% with SEQ ID NO: 7; and/or        -   one or more nucleotide sequence encoding a heterologous            glycerol dehydrogenase (E.C. 1.1.1.6) represented by SEQ ID            NO: 9 or a functional homologue of SEQ ID NO: 9 having            sequence identity of at least 60% with SEQ ID NO: 9-   Item 5. Cell according to item 4, wherein    -   c) is one or more nucleotide sequence encoding a heterologous        glycerol dehydrogenase (E.C. 1.1.1.6) represented by SEQ ID NO:        7 or a functional homologue of SEQ ID NO: 7 having sequence        identity of at least 60% with SEQ ID NO: 7.-   Item 6. Cell according to any of items 1 to 5, wherein    -   d) is one or more nucleotide sequence encoding a homologous or        heterologous dihydroxyacetone kinase (E.C. 2.7.1.28 or E.C.        2.7.1.29) represented by SEQ ID NO: 11 or a functional homologue        of SEQ ID NO: 11 having sequence identity of at least 60% with        SEQ ID NO: 11 and/or    -   one or more nucleotide sequence encoding a homologous or        heterologous dihydroxyacetone kinase (E.C. 2.7.1.28 or E.C.        2.7.1.29) represented by SEQ ID NO: 13 or a functional homologue        of SEQ ID NO: 13 having sequence identity of at least 60% with        SEQ ID NO: 13.-   Item 7. Cell according to item 6, wherein    -   d) is one or more nucleotide sequence encoding a homologous or        heterologous dihydroxyacetone kinase (E.C. 2.7.1.28 or E.C.        2.7.1.29) represented by SEQ ID NO: 13 or a functional homologue        of SEQ ID NO: 13 having sequence identity of at least 60% with        SEQ ID NO: 13.-   Item 8. Cell according to any of items 1 to 7 wherein    -   a) is one or more nucleotide sequence encoding a heterologous        NAD₊-dependent acetylating acetaldehyde dehydrogenase        represented by SEQ ID NO: 1 or a functional homologue of SEQ ID        NO: 1 having sequence identity of at least 60% with SEQ ID NO:        1; and/or        -   one or more nucleotide sequence encoding a heterologous            NAD₊-dependent acetylating acetaldehyde dehydrogenase            represented by SEQ ID NO: 2 or a functional homologue of SEQ            ID NO: 2 having sequence identity of at least 60% with SEQ            ID NO: 2 and/or        -   one or more nucleotide sequence encoding a heterologous            NAD₊-dependent acetylating acetaldehyde dehydrogenase            represented by SEQ ID NO: 3 or a functional homologue of SEQ            ID NO: 3 having sequence identity of at least 60% with SEQ            ID NO: 3.-   Item 9. Cell according to item 8 wherein    -   a) is one or more nucleotide sequence encoding a heterologous        NAD₊-dependent acetylating acetaldehyde dehydrogenase (E.C.        1.2.1.10) represented by SEQ ID NO: 1 or a functional homologue        of SEQ ID NO: 1 having sequence identity of at least 60% with        SEQ ID NO: 1; and/or        -   one or more nucleotide sequence encoding a heterologous            NAD₊-dependent acetylating acetaldehyde dehydrogenase (E.C.            1.2.1.10) represented by SEQ ID NO: 2 or a functional            homologue of SEQ ID NO: 2 having sequence identity of at            least 60% with SEQ ID NO: 2-   item 10. Cell according to item 9 wherein    -   a) is one or more nucleotide sequence encoding a heterologous        NAD₊-dependent acetylating acetaldehyde dehydrogenase        represented by SEQ ID NO: 2 or a functional homologue of SEQ ID        NO: 2 having sequence identity of at least 60% with SEQ ID NO:        2.-   Item 11. Cell according to any of item 1 to 10 wherein    -   a) is one or more nucleotide sequence encoding a heterologous        NAD₊-dependent acetylating acetaldehyde dehydrogenase        (E.C.1.2.1.10) represented by SEQ ID NO: 3 or a functional        homologue of SEQ ID NO: 3 having sequence identity of at least        60% with SEQ ID NO: 3;    -   b) is one or more nucleotide sequence encoding a homologous or        heterologous acetyl-CoA synthetase (E.C. 6.2.1.1) represented by        SEQ ID NO: 6 or a functional homologue of SEQ ID NO: 6 having        sequence identity of at least 60% with SEQ ID NO: 6;    -   c) is one or more nucleotide sequence encoding a heterologous        glycerol dehydrogenase (E.C. 1.1.1.6) represented by SEQ ID NO:        7 or a functional homologue of SEQ ID NO: 7 having sequence        identity of at least 60% with SEQ ID NO: 7; and    -   d) is one or more nucleotide sequence encoding a homologous or        heterologous dihydroxyacetone kinase (E.C. 2.7.1.28 or E.C.        2.7.1.29) represented by SEQ ID NO: 11 or a functional homologue        of SEQ ID NO: 11 having sequence identity of at least 60% with        SEQ ID NO: 11.-   Item 12. Cell according to any of item 1 to 10, wherein    -   a) is one or more nucleotide sequence encoding a heterologous        NAD₊-dependent acetylating acetaldehyde dehydrogenase (E.C.        1.2.1.10) represented by SEQ ID NO: 2 or a functional homologue        of SEQ ID NO: 2 having sequence identity of at least 60% with        SEQ ID NO: 2;    -   b) is one or more nucleotide sequence encoding a homologous or        heterologous acetyl-CoA synthetase (E.C. 6.2.1.1) represented by        SEQ ID NO: 6 or a functional homologue of SEQ ID NO: 6 having        sequence identity of at least 60% with SEQ ID NO: 6;    -   c) is one or more nucleotide sequence encoding a heterologous        glycerol 3-phosphate dehydrogenase (E.C. 1.1.1.8) represented by        SEQ ID NO: 9 or a functional homologue of SEQ ID NO: 9 having        sequence identity of at least 60% with SEQ ID NO: 9; and    -   d) is one or more nucleotide sequence encoding a homologous or        heterologous dihydroxyacetone kinase (E.C. 2.7.1.28 or E.C.        2.7.1.29) represented by SEQ ID NO: 11 or a functional homologue        of SEQ ID NO: 11 having sequence identity of at least 60% with        SEQ ID NO: 11.-   Item 13. Cell according to any of item 1 to 10, wherein a) is one or    more nucleotide sequence encoding a heterologous NAD₊-dependent    acetylating acetaldehyde dehydrogenase (E.C. 1.2.1.10) represented    by SEQ ID NO: 2 or a functional homologue of SEQ ID NO: 2 having    sequence identity of at least 60% with SEQ ID NO: 2;    -   b) is one or more nucleotide sequence encoding a homologous or        heterologous acetyl-CoA synthetase (E.C. 6.2.1.1) represented by        SEQ ID NO: 6 or a functional homologue of SEQ ID NO: 6 having        sequence identity of at least 60% with SEQ ID NO: 6;    -   c) is one or more nucleotide sequence encoding a heterologous        glycerol 3-phosphate dehydrogenase (E.C. 1.1.1.8) represented by        SEQ ID NO: 7 or a functional homologue of SEQ ID NO: 7 having        sequence identity of at least 60% with SEQ ID NO: 7; and    -   d) is one or more nucleotide sequence encoding a homologous or        heterologous dihydroxyacetone kinase (E.C. 2.7.1.28 or E.C.        2.7.1.29) represented by SEQ ID NO: 13 or a functional homologue        of SEQ ID NO: 13 having sequence identity of at least 60% with        SEQ ID NO: 13.-   Item 14. Cell according to any of item 1 to 10, wherein    -   a) is one or more nucleotide sequence encoding a heterologous        NAD₊-dependent acetylating acetaldehyde dehydrogenase (E.C.        1.2.1.10) represented by SEQ ID NO: 1 or a functional homologue        of SEQ ID NO: 1 having sequence identity of at least 60% with        SEQ ID NO: 1;    -   b) is one or more nucleotide sequence encoding a homologous or        heterologous acetyl-CoA synthetase (E.C. 6.2.1.1) represented by        SEQ ID NO: 6 or a functional homologue of SEQ ID NO: 6 having        sequence identity of at least 60% with SEQ ID NO: 6;    -   c) is one or more nucleotide sequence encoding a heterologous        glycerol 3-phosphate dehydrogenase (E.C. 1.1.1.8) represented by        SEQ ID NO: 7 or a functional homologue of SEQ ID NO: 7 having        sequence identity of at least 60% with SEQ ID NO: 7; and    -   d) is one or more nucleotide sequence encoding a homologous or        heterologous dihydroxyacetone kinase (E.C. 2.7.1.28 or E.C.        2.7.1.29) represented by SEQ ID NO: 13 or a functional homologue        of SEQ ID NO: 13 having sequence identity of at least 60% with        SEQ ID NO: 13.

The cell according to the invention may be prepared by modification of ahost cell, e.g. introduction of polynucleotides and/or expression ofproteins, e.g. nucleotides corresponding to the above features a) to d).

The polynucleotides or proteins may be homologous or heterologous to thegenome of the host cell. The term “heterologous”, with respect to thehost cell, means that the polynucleotide does not naturally occur in thegenome of the host cell or that the polypeptide is not naturallyproduced by that cell. “Homologous” with respect to a host cell, meansthat the polynucleotide does naturally occur in the genome of the hostcell or that the polypeptide is naturally produced by that cell.Homologous protein expression may e.g. be an overexpression orexpression of under the control a different promoter. Heterologousprotein expression involves expression of a protein that is notnaturally produced in the host cell.

The cell according to the invention is illustrated in FIG. 1. FIG. 1gives a schematic representation of the enzymatic reactions involved inthe conversion of glycerol and acetic acid (acetate) into ethanol.Acetate is first converted into acetyl-CoA through the yeast enzyme Acs(Acs1 and/or Acs2, encoded by the genes ACS1 and ACS2 respectively).Acetyl-CoA is then converted into acetaldehyde through the mhpF gene, ordirectly into ethanol through the bifunctional adhE enzyme from E. coli(or similar enzymes catalyzing the same conversion). Upon introductionof the glycerol consumption pathway, converting externally addedglycerol, an extra flow of NADH is generated. As indicated in FIG. 1,and in item 2 GPD1 and GPD2 genes may be deleted to avoid theintracellular production of glycerol and the utilization of NADH bythese enzymes.

The ethanol yield per consumed sugar (glucose or other sugars) increasesdue to elimination of glycerol production, ethanol generation fromacetate/acetic acid in the medium (and always present in lignocellulosichydrolysates) and glycerol externally added to the medium (orhydrolysate).

Throughout the present specification and the accompanying claims, thewords “comprise” and “include” and variations such as “comprises”,“comprising”, “includes” and “including” are to be interpretedinclusively. That is, these words are intended to convey the possibleinclusion of other elements or integers not specifically recited, wherethe context allows. The articles “a” and “an” are used herein to referto one or to more than one (i.e. to one or at least one) of thegrammatical object of the article. By way of example, “an element” maymean one element or more than one element.

“function” polypeptide”, is also designated herein as “polypeptide“function”” or “polypeptide”. “”function polypeptide polynucleotide”, isherein a polynucleotide that encodes the ““function” polypeptide. Theinvention further relates to a polynucleotide encoding such polypeptide,a nucleic acid construct comprising the polynucleotide encoding thepolypeptide and to a vector for the functional expression of aheterologous polypeptide in a (yeast) cell, said expression vectorcomprising a heterologous nucleic acid sequence operably linked to apromoter functional in the cell and said heterologous nucleic acidsequence encoding a polypeptide having the “function” enzymatic activityin (the cytosol of') said cell. A “function” polypeptide herein may haveone or more alternative and/or additional activities other than that ofthe “function” activity.

The E.C. codes mentioned herein are used only for clarification of a“function”, but should in no way be considered to be limiting to the“function”.

Any exogenous gene coding for an enzyme herein comprises a nucleotidesequence coding for an amino acid sequence with at least 50, 60, 65, 70,75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or100% amino acid sequence identity with any of SEQ ID's NO: X, whereinSEQ ID NO:X is any of the protein sequences in the sequence listing ofthis application. In particular for all of SEQ ID NO:'s 1-14. Theexogenous gene coding for an enzyme may also comprises a nucleotidesequence coding for an amino acid sequence having one or severalsubstitutions, insertions and/or deletions as compared to the amino acidsequence of any of SEQ ID NO: X. In particular for all of SEQ ID NO:'s1-14. Preferably the amino acid sequence has no more than 420, 380, 300,250, 200, 150, 100, 75, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1amino acid substitutions, insertions and/or deletions as compared to SEQID's NO: X. In particular for all of SEQ ID NO:'s 1-14.

Any exogenous gene coding for an enzyme herein comprises a nucleotidesequence with at least 40, 50, 60, 65, 70, 75, 80, 85, 86, 87, 88, 89,90, 95, 96, 97, 98, 99% or 100% nucleotide (DNA) sequence identity withany of SEQ ID's NO: Y, wherein SEQ ID NO: Y is any of the nucleotide(DNA) sequences in the sequence listing of this application. Inparticular for all of SEQ ID NO:'s 48-61.

The features a) to d) of item 1 will now be described in more detailbelow.

Feature a) one or more nucleotide sequence encoding a NAD₊-dependentacetylating acetaldehyde dehydrogenase:

The cell of the invention comprises an exogenous gene coding for anenzyme with the ability to reduce acetylCoA into acetaldehyde, whichgene confers to the cell the ability to convert acetylCoA (and/or aceticacid) into ethanol. An enzyme with the ability to reduce acetylCoA intoacetaldehyde is herein understood as a bifuntional enzyme whichcatalyzes the following reactions (adhE):

acetaldehyde+NADH⇄ethanol+NAD+  (1)

and/or

NAD++coenzyme A+acetaldehyde⇄NADH+acetyl-CoA   (2)

Thus, the enzyme catalyzes the conversion of acetaldehyde into ethanol(and vice versa) and is also referred to as an acetaldehydedehydrogenase (NAD₊-dependent). The enzyme is a bifunctional enzymewhich further catalyzes the conversion of coenzyme A and acetaldehydeinto acetyl-coA (and vice versa) also designated as acetaldehydedehydrogenase. This enzyme allows the re-oxidation of NADH whenacetyl-Coenzyme A is generated from acetate present in the growthmedium, and thereby glycerol synthesis is no longer needed for redoxcofactor balancing. The nucleic acid sequence encoding theNADH-dependent acetylating acetaldehyde dehydrogenase (E.C. 1.2.1.10)may in principle originate from any organism comprising a nucleic acidsequence encoding said dehydrogenase.

Known NAD₊-dependent acetylating acetaldehyde dehydrogenases that cancatalyse the NADH-dependent reduction of acetyl-Coenzyme A toacetaldehyde may in general be divided in three types of NADH-dependentacetylating acetaldehyde dehydrogenase functional homologues:

-   1) Bifunctional proteins that catalyse the reversible conversion of    acetyl-Coenzyme A to acetaldehyde, and the subsequent reversible    conversion of acetaldehyde to ethanol. An example of this type of    proteins is the AdhE protein in E. coli (Gen Bank No: NP-415757).    AdhE appears to be the evolutionary product of gene fusion. The    NH2-terminal region of the AdhE protein is highly homologous to    aldehyde:NADH oxidoreductases, whereas the COOH-terminal region is    homologous to a family of Fe2+-dependent ethanol:NADH    oxidoreductases (Membrillo-Hernandez et al., (2000) J. Biol. Chem.    275: 33869-33875). The E. coli AdhE is subject to metal-catalyzed    oxidation and therefore oxygen-sensitive (Tamarit et al. (1998) J.    Biol. Chem. 273:3027-32).-   2) Proteins that catalyse the reversible conversion of    acetyl-Coenzyme A to acetaldehyde in strictly or facultative    anaerobic micro-organisms but do not possess alcohol dehydrogenase    activity. An example of this type of proteins has been reported in    Clostridium kluyveri (Smith et al. (1980) Arch. Biochem. Biophys.    203: 663-675). An acetylating acetaldehyde dehydrogenase has been    annotated in the genome of Clostridium kluyveri DSM 555 (GenBank No:    EDK33116). A homologous protein AcdH is identified in the genome of    Lactobacillus plantarum (GenBank No: NP-784141). Another example of    this type of proteins is the said gene product in Clostridium    beijerinckii NRRL B593 (Toth et al. (1999) Appl. Environ. Microbiol.    65: 4973-4980, GenBank No: AAD31841).-   3) Proteins that are part of a bifunctional aldolase-dehydrogenase    complex involved in 4-hydroxy-2-ketovalerate catabolism. Such    bifunctional enzymes catalyze the final two steps of the    meta-cleavage pathway for catechol, an intermediate in many    bacterial species in the degradation of phenols, toluates,    naphthalene, biphenyls and other aromatic compounds (Powlowski and    Shingler (1994) Biodegradation 5, 219-236).    4-Hydroxy-2-ketovaleraties first converted by    4-hydroxy-2-ketovalerate aldolase to pyruvate and acetaldehyde,    subsequently acetaldehyde is converted by acetylating acetaldehyde    dehydrogenase to acetyl-CoA. An example of this type of acetylating    acetaldehyde dehydrogenase is the DmpF protein in Pseudomonas sp    CF600 (GenBank No: CAA43226) (Shingler et al. (1992) J. Bacteriol.    174:71 1-24). The E. coli MphF protein (Ferrandez et al. (1997) J.    Bacteriol. 179: 2573-2581, GenBank No: NP-414885) is homologous to    the DmpF protein in Pseudomonas sp. CF600.

A suitable nucleic acid sequence may in particular be found in anorganism selected from the group of Escherichia, in particular E. coli;Mycobacterium, in particular Mycobacterium marinum, Mycobacteriumulcerans, Mycobacterium tuberculosis; Carboxydothermus, in particularCarboxydothermus hydrogenoformans; Entamoeba, in particular Entamoebahistolytica; Shigella, in particular Shigella sonnei; Burkholderia, inparticular Burkholderia pseudomallei, Klebsiella, in particularKlebsiella pneumoniae; Azotobacter, in particular Azotobacteruinelandii; Azoarcus sp; Cupriauidus, in particular Cupriauidustaiwanensis; Pseudomonas, in particular Pseudomonas sp. CF600;Pelomaculum, in particular Pelotomaculum thermopropionicum. Preferably,the nucleic acid sequence encoding the NADH-dependent acetylatingacetaldehyde dehydrogenase originates from Escherichia, more preferablyfrom E. coli.

Particularly suitable is an mhpF gene from E. coli, or a functionalhomologue thereof. This gene is described in Ferrandez et al. (1997) J.Bacteriol. 179:2573-2581. Good results have been obtained with S.cerevisiae, wherein an mhpF gene from E. coli has been incorporated.

In a further advantageous embodiment the nucleic acid sequence encodingan (acetylating) acetaldehyde dehydrogenase is from, in particularPseudomonas dmpF from Pseudomonas sp. CF600.

In principle, the nucleic acid sequence encoding the NAD₊-dependent,acetylating acetaldehyde dehydrogenase may be a wild type nucleic acidsequence. A preferred nucleic acid sequence encodes the NAD₊-dependent,acetylating acetaldehyde dehydrogenase represented by SEQ ID NO: 2, SEQID NO: 29 in WO2011010923, or a functional homologue of SEQ ID NO: 2 orSEQ ID NO: 29 in WO2011010923. In particular the nucleic acid sequencecomprises a sequence according to SEQ ID NO: 1. SEQ ID NO: 28 inWO2011010923 or a functional homologue of SEQ ID NO: 1 or SEQ ID NO: 28in WO2011010923.

Further, an acetylating acetaldehyde dehydrogenase (or nucleic acidsequence encoding such activity) may in for instance be selected fromthe group of Escherichia coli adhE, Entamoeba histolytica adh2,Staphylococcus aureus adhE, Piromyces sp. E2 adhE, Clostridium kluyveriEDK33116, Lactobacillus plantarum acdH, and Pseudomonas putida YP001268189. For sequences of these enzymes, nucleic acid sequencesencoding these enzymes and methodology to incorporate the nucleic acidsequence into a host cell, reference is made to WO 20091013159, inparticular Example 3, Table 1 (page 26) and the Sequence ID numbersmentioned therein, of which publication Table 1 and the sequencesrepresented by the Sequence ID numbers mentioned in said Table areincorporated herein by reference.

It is further understood, that in a preferred embodiment, that the cellhas endogenous alcohol dehydrogenase activities which allow the cell,being provided with acetaldehyde dehydrogenase activity, to complete theconversion of acetyl-CoA into ethanol. It is further also preferred thatthe host cell has endogenous acetyl-CoA synthetase which allow the cell,being provided with acetaldehyde dehydrogenase activity, to complete theconversion of acetic acid (via acetyl-CoA) into ethanol.

The exogenous gene coding for an enzyme with acetaldehyde dehydrogenaseactivity preferably comprises a nucleotide sequence coding for an aminoacid sequence with at least 40, 50, 60, 65, 70, 75, 80, 85, 90, 95, 98,99% amino acid sequence identity with any of SEQ ID's NO: 1 to SEQ IDNO: 5, preferably of SEQ ID NO: 1 to SEQ ID NO: 3, more preferably ofSEQ ID NO: 1 or SEQ ID NO: 2. The exogenous gene coding for an enzymewith acetaldehyde dehydrogenase activity may also comprises a nucleotidesequence coding for an amino acid sequence having one or severalsubstitutions, insertions and/or deletions as compared to the amino acidsequence of any of SEQ ID's NO: 1 to SEQ ID NO: 5, preferably of SEQ IDNO: 1 to SEQ ID NO: 3, more preferably of SEQ ID NO: 1 or SEQ ID NO: 2.Preferably the amino acid sequence has no more than 420, 380, 300, 250,200, 150, 100, 75, 50, 40, 30, 20, 10 or 5 amino acid substitutions,insertions and/or deletions as compared to SEQ ID's NO: 1 to SEQ ID NO:5 respectively, preferably to SEQ ID NO: 1 to SEQ ID NO: 3 respectively,more preferably to SEQ ID NO: 1 or SEQ ID NO: 2 respectively.

The exogenous gene coding for an enzyme with acetaldehyde dehydrogenaseherein comprises a nucleotide sequence with at least 40, 50, 60, 65, 70,75, 80, 85, 86, 87, 88, 89, 90, 95, 96, 97, 98, 99% or 100% nucleotide(DNA) sequence identity with any of SEQ ID's NO: Y, wherein SEQ ID NO: Yis any of the nucleotide (DNA) sequences 48-52.

Some organisms that could be a source of adhE enzymes that may besuitable for expression in the cell of the invention are mentioned intable 1.

TABLE 1 Organism with enzymes with alcohol/ acetaldehyde dehydrogenase(adhE) activity Organism Piromyces sp E2 Arthrospira platensisSynechococcus sp. Microcystis aeruginosa Microcoleus chthonoplastesLyngbya sp. Thermosynechococcus elongatus Treponema phagedenisClostridium difficile Clostridium carboxidivorans Clostridiumacetobutylicum

Examples of suitable enzymes are adhE of Escherichia coli, acdH ofLactobacillus plantarum, eutE of Escherichia coli, Lin1129 of Listeriainnocua and adhE from Staphylococcus aureus. See below tables 2(a) to2(e) for BLAST of these enzymes, giving suitable alternativealcohol/acetaldehyde dehydrogenases that are tested in the examplesbelow.

TABLE 2(a) BLAST Query - adHE from Escherichia coli Identity AccessionDescription (%) number bifunctional acetaldehyde-CoA/alcohol 100NP_309768.1 dehydrogenase [Escherichia coli O157:H7 str. Sakai]bifunctional acetaldehyde-CoA/alcohol 99 YP_540449.1 dehydrogenase[Escherichia coli UTI89] bifunctional acetaldehyde-CoA/alcohol 95YP_001177024.1 dehydrogenase [Enterobacter sp. 638]

TABLE 2(b) BLAST Query - acdH from Lactobacillus plantarum IdentityAccession Description (%) number acetaldehyde dehydrogenase 100YP_004888365.1 [Lactobacillus plantarum WCFS1] acetaldehydedehydrogenase 95 CCC16763.1 [Lactobacillus pentosus IG1]aldehyde-alcohol dehydrogenase 58 WP_016251441.1 [Enterococcus cecorum]aldehyde-alcohol dehydrogenase 2 57 WP_016623694.1 [Enterococcusfaecalis] bifunctional acetaldehyde-CoA/alcohol 55 WP_010493695.1dehydrogenase [Lactobacillus zeae] alcohol dehydrogenase 54WP_003280110.1 [Bacillus thuringiensis] bifunctionalacetaldehyde-CoA/alcohol 53 WP_009931954.1 dehydrogenase, partial[Listeria monocytogenes]

TABLE 2(c) BLAST Query - eutE from Escherichia coli Identity AccessionDescription (%) number aldehyde oxidoreductase, ethanolamine 100NP_416950.1 utilization protein [Escherichia coli str. K-12 substr.MG1655] ethanolamine utilization; acetaldehyde 99 NP_289007.1dehydrogenase [Escherichia coli O157:H7 str. EDL933] aldehydedehydrogenase 99 WP_001075674.1 [Escherichia albertii]

TABLE 2(d) BLAST Query - Lin1129 from Listeria innocua IdentityAccession Description (%) number aldehyde dehydrogenase 100 NP_470466.1[Listeria innocua] >emb|CAC96360.1| lin1129 [Listeria innocua Clip11262]ethanolamine utilization protein EutE 99 WP_003761764.1 [Listeriainnocua] aldehyde dehydrogenase 95 AGR09081.1 [Listeria monocytogenes]hypothetical protein 64 WP_010739890.1 [Enterococcus malodoratus]aldehyde dehydrogenase 59 WP_004699364.1 [Yersinia aldovae] aldehydedehydrogenase EutE 58 WP_004205473.1 [Klebsiella pneumoniae]

TABLE 2(e) BLAST Query - adhE from Staphylococcus aureus IdentityAccession Description (%) number bifunctional acetaldehyde-CoA/alcohol100 NP_370672.1 dehydrogenase [Staphylococcus aureus subsp. aureus Mu50]aldehyde dehydrogenase family protein 99 YP_008127042.1 [Staphylococcusaureus CA-347] bifunctional acetaldehyde-CoA/alcohol 85 WP_002495347.1dehydrogenase [Staphylococcus epidermidis] aldehyde-alcoholdehydrogenase 2 75 WP_016623694.1 [Enterococcus faecalis]

Feature b: one or more nucleotide sequence encoding a acetyl-CoAsynthetase (E.C. 6.2.1.1);

Acetyl-CoA synthetase (also known as acetate-CoA ligase andacetyl-activating enzyme) is a ubiquitous enzyme, found in bothprokaryotes and eukaryotes, which catalyses the formation of acetyl-CoAfrom acetate, coenzyme A (CoA) and ATP as shown below [PMID: 15316652]:

ATP+acetate+CoA=AMP+diphosphate+acetyl-CoA   (4)

The activity of this enzyme is crucial for maintaining the requiredlevels of acetyl-CoA, a key intermediate in many important biosyntheticand catabolic processes. It is especially important in eukayotic speciesas it is the only route for the activation of acetate to acetyl-CoA inthese organisms (some prokaryotic species can also activate acetate byeither acetate kinase/phosphotransacetylase or by ADP-forming acetyl-CoAsynthase). Eukaryotes typically have two isoforms of acetyl-CoAsynthase, a cytosolic form involved in biosynthetic processes and amitochondrial form primarily involved in energy generation.

The crystal structures of a eukaryotic (e.g. from yeast) and bacterial(e.g. from Salmonella) form of this enzyme have been determined. Theyeast enzyme is trimeric, while the bacterial enzyme is monomeric. Thetrimeric state of the yeast protein may be unique to this organismhowever, as the residues involved in the trimer interface are poorlyconserved in other sequences. Despite differences in the oligomericstate of the two enzyme, the structures of the monomers are almostidentical. A large N-terminal domain (˜500 residues) containing twoparallel beta sheets is followed by a small (˜110 residues) C-terminaldomain containing a three-stranded beta sheet with helices. The activesite occurs at the domain interface, with its contents determining theorientation of the C-terminal domain.

When the cell is a yeast cell the endogenous ACS are preferred accordingto the invention, in an embodiment they are overexpressed in yeast cell.

Examples of suitable are listed in table 3. At the top of table 3 theACS2 used in the examples and that is BLASTED is mentioned.

TABLE 3 BLAST Query - ACS2 from Saccharomyces cerevisiae IdentityAccession Description (%) number acetate--CoA ligase ACS2 100NP_013254.1 [Saccharomyces cerevisiae S288c] acetyl CoA synthetase 99EDN59693.1 [Saccharomyces cerevisiae YJM789] acetate--CoA ligase 85XP_453827.1 [Kluyveromyces lactis NRRL Y-1140] acetate--CoA ligase 83XP_445089.1 [Candida glabrata CBS 138] acetate--CoA ligase 68XP_001385819.1 [Scheffersomyces stipitis CBS 6054] acetyl-coenzyme Asynthetase FacA 63 EDP50475.1 [Aspergillus fumigatus A1163] acetate--CoAligase facA-Penicillium 62 XP_002564696.1 chrysogenum [Penicilliumchrysogenum Wisconsin 54-1255]The exogenous gene coding for an enzyme with ACS activity preferablycomprises a nucleotide sequence coding for an amino acid sequence withat least 50, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99% amino acid sequenceidentity with SEQ ID NO: 6. The exogenous gene coding for an enzyme withACS activity may also comprises a nucleotide sequence coding for anamino acid sequence having one or several substitutions, insertionsand/or deletions as compared to the amino acid sequence of SEQ ID's NO:6. Preferably the amino acid sequence has no more than 420, 380, 300,250, 200, 150, 100, 75, 50, 40, 30, 20, 10 or 5 amino acidsubstitutions, insertions and/or deletions as compared to SEQ ID's NO:6.

The exogenous gene coding for an enzyme with ACS activity hereincomprises a nucleotide sequence with at least 40, 50, 60, 65, 70, 75,80, 85, 86, 87, 88, 89, 90, 95, 96, 97, 98, 99% or 100% nucleotide (DNA)sequence identity with SEQ ID's NO: 53.

Feature c): According to feature c), the cell comprises one or morenucleotide sequence encoding a glycerol dehydrogenase (E.C. 1.1.1.6).Glycerol dehydrogenase (EC 1.1.1.6) is an enzyme that catalyzes thechemical reaction

glycerol+NAD⁺

glycerone+NADH+H⁺  (5)

Thus, the two substrates of this enzyme are glycerol and NAD⁺, whereasits three products are glycerone, NADH, and H⁺. Glyceron anddihydroxyacetone are herein synonyms.

This enzyme belongs to the family of oxidoreductases, specifically thoseacting on the CH—OH group of donor with NAD⁺ or NADP⁺ as acceptor. Thesystematic name of this enzyme class is glycerol:NAD⁺ 2-oxidoreductase.Other names in common use include glycerin dehydrogenase, andNAD⁺-linked glycerol dehydrogenase. This enzyme participates inglycerolipid metabolism. Structural studies have shown that the enzymeis zinc-dependent with the active site lying between the two domains ofthe protein.

Examples of suitable glycerol dehydrogenases are listed in table 4(a) to4(d). At the top of each table the gldA used in the examples and that isBLASTED is mentioned.

TABLE 4(a) BLAST Query - gldA from Escherichia coli Identity AccessionDescription (%) number glycerol dehydrogenase, NAD 100 NP_418380.4[Escherichia coli str. K-12 substr. MG1655] glycerol dehydrogenase 99YP_002331714.1 [Escherichia coli O127:H6 str. E2348/69] glyceroldehydrogenase 94 WP_006686227.1 [Citrobacter youngae] glyceroldehydrogenase 92 WP_003840533.1 [Citrobacter freundii]

TABLE 4(b) BLAST Query - gldA from Klebsiella pneumoniae IdentityAccession Description (%) number glycerol dehydrogenase 100YP_002236495.1 [Klebsiella pneumoniae 342] glycerol dehydrogenase 93WP_003024745.1 [Citrobacter freundii] Glycerol dehydrogenase (EC1.1.1.6) 92 YP_004590977.1 [Enterobacter aerogenes EA1509E] glyceroldehydrogenase 91 WP_016241524.1 [Escherichia coli] glyceroldehydrogenase 87 See examples [Enterococcus aerogenes] herein strainswith CAS15 glycerol dehydrogenase 74 WP_004701845.1 [Yersinia aldovae]glycerol dehydrogenase 61 WP_017375113.1 [Enterobacteriaceae bacteriumLSJC7] glycerol dehydrogenase 60 WP_006686227.1 [Citrobacter youngae]

TABLE 4(c) BLAST Query - gldA from Enterococcus aerogenes IdentityAccession Description (%) number glycerol dehydrogenase 100YP_004591726.1 [Enterobacter aerogenes KCTC 2190] Glycerol dehydrogenase(EC 1.1.1.6) 99 YP_007390021.1 [Enterobacter aerogenes EA1509E] glyceroldehydrogenase 92 WP_004203683.1 [Klebsiella pneumoniae] glyceroldehydrogenase 88 WP_001322519.1 [Escherichia coli] See examples hereinstrains with CAS13 glycerol dehydrogenase 87 YP_003615506.1[Enterobacter cloacae subsp. cloacae ATCC 13047]

TABLE 4(d) BLAST Query - gldA from Yersinia aldovae Identity AccessionDescription (%) number glycerol dehydrogenase 100 WP_004701845.1[Yersinia aldovae] glycerol dehydrogenase 95 WP_005189747.1 [Yersiniaintermedia] glycerol dehydrogenase 81 YP_008232202.1 [Serratialiquefaciens ATCC 27592] glycerol dehydrogenase 76 WP_016241524.1[Escherichia coli] See examples herein strains with CAS13. hypotheticalprotein EAE_03845 75 YP_004590977.1 [Enterobacter aerogenes KCTC 2190]glycerol dehydrogenase 65 WP_017410769.1 [Aeromonas hydrophila]

The exogenous gene coding for an enzyme with gldA activity preferablycomprises a nucleotide sequence coding for an amino acid sequence withat least 50, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99% amino acid sequenceidentity with any of SEQ ID's NO: 7 to SEQ ID NO: 10, preferably of SEQID NO:7 or SEQ ID NO: 9. The exogenous gene coding for an enzyme withacetaldehyde dehydrogenase activity may also comprises a nucleotidesequence coding for an amino acid sequence having one or severalsubstitutions, insertions and/or deletions as compared to the amino acidsequence of any of SEQ ID's NO: 7 to SEQ ID NO: 10, preferably of SEQ IDNO:7 or SEQ ID NO: 9. Preferably the amino acid sequence has no morethan 300, 250, 200, 150, 100, 75, 50, 40, 30, 20, 10 or 5 amino acidsubstitutions, insertions and/or deletions as compared to SEQ ID's NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO: 10 respectively, preferably toSEQ ID NO:7 or SEQ ID NO: 9 respectively.

The exogenous gene coding for an enzyme with gldA activity hereincomprises a nucleotide sequence with at least 40, 50, 60, 65, 70, 75,80, 85, 86, 87, 88, 89, 90, 95, 96, 97, 98, 99% or 100% nucleotide (DNA)sequence identity with any of SEQ ID's NO: 54-57.

Feature d): one or more heterologous nucleotide sequence encoding adihydroxyacetone kinase (E.C. 2.7.1.28 or E.C. 2.7.1.29), Thedihydroxyacetone kinase enzyme is involved in reactions:

Glycerone=dihydroxyacetone.

This family consists of examples of the single chain form ofdihydroxyacetone kinase (also called glycerone kinase) that uses ATP (EC2.7.1.29 or EC 2.7.1.28) as the phosphate donor, rather than aphosphoprotein as in Escherichia coli. This form has separable domainshomologous to the K and L subunits of the E. coli enzyme, and is foundin yeasts and other eukaryotes and in some bacteria, includingCitrobacter freundii. The member from tomato has been shown tophosphorylate dihydroxyacetone, 3,4-dihydroxy-2-butanone, and some otheraldoses and ketoses. Members from mammals have been shown to catalyseboth the phosphorylation of dihydroxyacetone and the splitting ofribonucleoside diphosphate-X compounds among which FAD is the bestsubstrate. In yeast there are two isozymes of dihydroxyacetone kinase(Dak1 and Dak2). When the cell is a yeast cell the endogenous DAK's arepreferred according to the invention, in an embodiment they areoverexpressed in yeast cell.

Examples of suitable dihydroxyacetone kinases are listed in table 5(a)to 5(d). At the top of each table the DAK's used in the examples andthat is BLASTED is mentioned.

TABLE 5(a) BLAST Query - DAK1 from Saccharomyces cerevisiae IdentityAccession Description (%) number Dak1p 100 NP_013641.1 [Saccharomycescerevisiae S288c] dihydroxyacetone kinase 99 EDN64325.1 [Saccharomycescerevisiae YJM789] DAK1-like protein 95 EJT44075.1 [Saccharomyceskudriavzevii IFO 1802] ZYBA0S11-03576g1_1 77 CDF91470.1[Zygosaccharomyces bailii CLIB 213] hypothetical protein 70 XP_451751.1[Kluyveromyces lactis NRRL Y-1140] hypothetical protein 63 XP_449263.1[Candida glabrata CBS 138] Dak2p 44 NP_116602.1 [Saccharomycescerevisiae S288c] DAK1 41 See examples [Yarrowia lipolytica] hereinstrains with CAS23

TABLE 5(b) BLAST Query - dhaK from Klebsiella pneumoniae IdentityAccession Description (%) number dihydroxyacetone kinase subunit DhaK100 YP_002236493.1 [Klebsiella pneumoniae 342] dihydroxyacetone kinasesubunit K 99 WP_004149886.1 [Klebsiella pneumoniae] dihydroxyacetonekinase subunit K 96 WP_020077889.1 [Enterobacter aerogenes]dihydroxyacetone kinase subunit DhaK 88 YP_002407536.1 [Escherichia coliIAI39] dihydroxyacetone kinase, DhaK subunit 87 WP_001398949.1[Escherichia coli]

TABLE 5(c) BLAST Query - DAK1 from Yarrowia lipolytica IdentityAccession Description (%) number YALI0F09273p 100 XP_505199.1 [Yarrowialipolytica] dihydroxyacetone kinase 46 AAC83220.1 [Schizosaccharomycespombe] dihydroxyacetone kinase Dak1 45 NP_593241.1 [Schizosaccharomycespombe 972h-] dihydroxyacetone kinase 44 EDV12567.1 [Saccharomycescerevisiae RM11-1a] Dak2p 44 EEU04233.1 [Saccharomyces cerevisiaeJAY291] BN860_19306g1_1 44 CDF87998.1 [Zygosaccharomyces bailii CLIB213] Dak1p 42 EIW08612.1 [Saccharomyces cerevisiae CEN.PK113-7D] Seeexamples herein strains with CAS21

TABLE 5(d) BLAST Query-DAK1 from Schizosaccharomyces pombe IdentityAccession Description (%) number dihydroxyacetone kinase Dak1 100NP_593241.1 [Schizosaccharomyces pombe 972h-] putative dihydroxyacetonekinase protein 48 EMR88164.1 [Botryotinia fuckeliana BcDW1]Dihydroxyacetone kinase 1 48 ENH64704.1 [Fusarium oxysporum f. sp.cubense race 1] Dak1p 46 EIW08612.1 [Saccharomyces cerevisiaeCEN.PK113-7D] Dak2p 44 EEU04233.1 [Saccharomyces cerevisiae JAY291]dihydroxyacetone kinase 42 EHY55064.1 [Exophiala dermatitidisNIH/UT8656]

The exogenous gene coding for an enzyme with DAK activity preferablycomprises a nucleotide sequence coding for an amino acid sequence withat least 40, 50, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99% amino acidsequence identity with any of SEQ ID's NO: 11 to SEQ ID NO: 14,preferably SEQ ID NO: 11 or SEQ ID NO: 13. The exogenous gene coding foran enzyme with acetaldehyde dehydrogenase activity may also comprises anucleotide sequence coding for an amino acid sequence having one orseveral substitutions, insertions and/or deletions as compared to theamino acid sequence of any of SEQ ID's NO: 11 to SEQ ID NO: 14,preferably SEQ ID NO: 11 or SEQ ID NO: 13. Preferably the amino acidsequence has no more than 420, 380, 300, 250, 200, 150, 100, 75, 50, 40,30, 20, 10 or 5 amino acid substitutions, insertions and/or deletions ascompared to SEQ ID NO: 11, SEQ NO: 12, SEQ ID NO; 13 or SEQ ID NO: 14respectively, preferably to SEQ ID NO: 11 or SEQ ID NO: 13 respectively.

The exogenous gene coding for an enzyme with DAK activity hereincomprises a nucleotide sequence with at least 40, 50, 60, 65, 70, 75,80, 85, 86, 87, 88, 89, 90, 95, 96, 97, 98, 99% or 100% nucleotide (DNA)sequence identity with any of SEQ ID's NO: 58-61.

In an embodiment, the cell comprises a deletion or disruption of one ormore endogenous nucleotide sequence encoding a glycerol 3-phosphatephosphohydrolase and/or encoding a glycerol 3-phosphate dehydrogenasegene:

Herein in the cell, enzymatic activity needed for the NADH-dependentglycerol synthesis is reduced or deleted. The reduction or deleted ofthis enzymatic activity can be achieved by modifying one or more genesencoding a NAD-dependent glycerol 3-phosphate dehydrogenase activity(GPD) or one or more genes encoding a glycerol phosphate phosphataseactivity (GPP), such that the enzyme is expressed considerably less thanin the wild-type or such that the gene encoded a polypeptide withreduced activity.

Such modifications can be carried out using commonly knownbiotechnological techniques, and may in particular include one or moreknock-out mutations or site-directed mutagenesis of promoter regions orcoding regions of the structural genes encoding GPD and/or GPP.Alternatively, yeast strains that are defective in glycerol productionmay be obtained by random mutagenesis followed by selection of strainswith reduced or absent activity of GPD and/or GPP. S. cerevisiae GPD1,GPD2, GPP1 and GPP2 genes are shown in WO2011010923, and are disclosedin SEQ ID NO: 24-27 of that application.

Preferably at least one gene encoding a GPD or at least one geneencoding a GPP is entirely deleted, or at least a part of the gene isdeleted that encodes a part of the enzyme that is essential for itsactivity. In particular, good results have been achieved with a S.cerevisiae cell, wherein the open reading frames of the GPD1 gene and ofthe GPD2 gene have been inactivated. Inactivation of a structural gene(target gene) can be accomplished by a person skilled in the art bysynthetically synthesizing or otherwise constructing a DNA fragmentconsisting of a selectable marker gene flanked by DNA sequences that areidentical to sequences that flank the region of the host cell's genomethat is to be deleted. In particular, good results have been obtainedwith the inactivation of the GPD1 and GPD2 genes in Saccharomycescerevisiae by integration of the marker genes kanMX and hphMX4.Subsequently this DNA fragment is transformed into a host cell.Transformed cells that express the dominant marker gene are checked forcorrect replacement of the region that was designed to be deleted, forexample by a diagnostic polymerase chain reaction or Southernhybridization.

Thus, in the cells of the invention, the specific glycerol 3-phosphatephosphohydrolase and/or encoding a glycerol 3-phosphate dehydrogenasegene is reduced. In the cells of the invention, the specificglycerolphosphate dehydrogenase activity is preferably reduced by atleast a factor 0.8, 0.5, 0.3, 0.1, 0.05 or 0.01 as compared to a strainwhich is genetically identical except for the genetic modificationcausing the reduction in specific activity, preferably under anaerobicconditions. Glycerolphosphate dehydrogenase activity may be determinedas described by Overkamp et al. (2002, Yeast 19:509-520).

Preferably, the genetic modifications reduce or inactivate theexpression of each endogenous copy of the gene encoding a specificglycerolphosphate dehydrogenase in the cell's genome. A given cell maycomprise multiple copies of the gene encoding a specificglycerolphosphate dehydrogenase with one and the same amino acidsequence as a result of di-, poly- or aneuploidy. In such instancespreferably the expression of each copy of the specific gene that encodesthe glycerolphosphate dehydrogenase is reduced or inactivated.Alternatively, a cell may contain several different (iso)enzymes withglycerolphosphate dehydrogenase activity that differ in amino acidsequence and that are each encoded by a different gene. In suchinstances, in some embodiments of the invention it may be preferred thatonly certain types of the isoenzymes are reduced or inactivated whileother types remain unaffected. Preferably, however, expression of allcopies of genes encoding (iso)enzymes with glycerolphosphatedehydrogenase activity is reduced or inactivated.

Preferably, a gene encoding glycerolphosphate dehydrogenase activity isinactivated by deletion of at least part of the gene or by disruption ofthe gene, whereby in this context the term gene also includes anynon-coding sequence up- or down-stream of the coding sequence, the(partial) deletion or inactivation of which results in a reduction ofexpression of glycerolphosphate dehydrogenase activity in the host cell.

A preferred gene encoding a glycerolphosphate dehydrogenase whoseactivity is to be reduced or inactivated in the cell of the invention isthe S. cerevisiae GPD1 as described by van den Berg and Steensma (1997,Yeast 13:551-559), encoding the amino acid sequence GPD1 and orthologuesthereof in other species.

Suitable examples of organisms (hosts) comprising an enzyme withglycerolphosphate dehydrogenase activity belonging to the genusSaccharomyces, Naumovozyna, Candida Vanderwaltozyma andZygosaccharomyces are provided in Table 6.

TABLE 6 Enzymes with glycerolphosphate dehydrogenase (GPD1) activityOrganism Amino acid identity (%) S. cerevisiae 100%  Naumovozymadairenensis 79% Naumovozyma castellii 80% Candida glabrata 77%Vanderwaltozyma polyspora 77% Zygosaccharomyces rouxii 74%Saccharomycopsis fibuligera 61%

However, in some strains e.g. of Saccharomyces, Candida andZygosaccharomyces a second gene encoding a glycerolphosphatedehydrogenase is active, i.e. the GPD2, see e.g. Overkamp et al. (2002,supra). Another preferred gene encoding a glycerolphosphatedehydrogenase whose activity is to be reduced or inactivated in the cellof the invention therefore is an S. cerevisiae GPD2 as described byOverkamp et al. (2002, supra), encoding the amino acid sequence GPD2 andorthologues thereof in other species.

Suitable examples of organisms (hosts) comprising an enzyme withglycerolphosphate dehydrogenase activity belonging to the genus (Zygo)Saccharomyces and Candida are provided in Table 7.

TABLE 7 Enzymes with glycerol phosphate dehydrogenase (GPD2) activityOrganism Amino acid identity (%) S. cerevisiae 100%  Candida glabrata75% Zygosaccharomyces rouxii 73% Spathaspora passalidarum 62%Scheffersomyces stipitis 61%

In an embodiment, the cell is a yeast wherein the genome of the yeastcell comprises a mutation in at least one gene selected from the groupof GPD1, GPD2, GPP1 and GPP2, which mutation may be a knock-outmutation, which knock-out mutation may be a complete deletion of atleast one of said genes in comparison to the yeast cell's correspondingwild-type yeast gene.

To increase the likelihood that the enzyme activities herein areexpressed at sufficient levels and in active form in the transformedhost cells of the invention, the nucleotide sequence encoding theseenzymes, and other enzymes of the invention (see below), are preferablyadapted to optimise their codon usage to that of the host cell inquestion. The adaptiveness of a nucleotide sequence encoding an enzymeto the codon usage of a host cell may be expressed as codon adaptationindex (CAI). The codon adaptation index is herein defined as ameasurement of the relative adaptiveness of the codon usage of a genetowards the codon usage of highly expressed genes in a particular hostcell or organism. The relative adaptiveness (w) of each codon is theratio of the usage of each codon, to that of the most abundant codon forthe same amino acid. The CAI index is defined as the geometric mean ofthese relative adaptiveness values. Non-synonymous codons andtermination codons (dependent on genetic code) are excluded. CAI valuesrange from 0 to 1, with higher values indicating a higher proportion ofthe most abundant codons (see Sharp and Li, 1987, Nucleic Acids Research15: 1281-1295; also see: Jansen et al., 2003, Nucleic Acids Res.31(8):2242-51). An adapted nucleotide sequence preferably has a CAI ofat least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9. Most preferred arethe sequences which have been codon optimised for expression in thefungal host cell in question such as e.g. S. cerevisiae cells.

Strain construction. The strain construction approach used herein in theexamples is described in patent application PCT/EP2013/056623. Itdescribes the techniques enabling the construction of expressioncassettes from various genes of interest in such a way, that thesecassettes are combined into a pathway and integrated in a specific locusof the yeast genome upon transformation of this yeast.

An overview of strain construction approach used in the examples isgiven in FIG. 2. FIG. 2 shows a schematic display of the strainconstruction approach. INT (integration) flanks and expression cassettes(CAS), including selectable marker, are amplified using PCR andtransferred into yeast. Recombination will take place between theconnectors (designated 5, a, b, c and 3 respectively in FIG. 2)resulting in the integration of the pathway in the desired location inthe yeast genome (in this case, INT1). The number of genes of interestmay be extended, as described in the examples. Unique connectors wereused to facilitate recombination of the separate expression cassettesand integration into the genome of the recipient cell. Any other strainconstruction methods according to the prior art may equally be used toconstruct the strains of the invention.

Homology & Identity.

Amino acid or nucleotide sequences are said to be homologous whenexhibiting a certain level of similarity. Two sequences being homologousindicate a common evolutionary origin. Whether two homologous sequencesare closely related or more distantly related is indicated by “percentidentity” or “percent similarity”, which is high or low respectively.Although disputed, to indicate “percent identity” or “percentsimilarity”, “level of homology” or “percent homology” is frequentlyused interchangeably.

A comparison of sequences and determination of percent identity betweentwo sequences can be accomplished using a mathematical algorithm. Theskilled person will be aware of the fact that several different computerprograms are available to align two sequences and determine the homologybetween two sequences (Kruskal, J. B. (1983) An overview of sequencecomparison In D. Sankoff and J. B. Kruskal, (ed.), Time warps, stringedits and macromolecules: the theory and practice of sequencecomparison, pp. 1-44 Addison Wesley). The percent identity between twoamino acid sequences can be determined using the Needleman and Wunschalgorithm for the alignment of two sequences. (Needleman, S. B. andWunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). The algorithm alignsamino acid sequences as well as nucleotide sequences. TheNeedleman-Wunsch algorithm has been implemented in the computer programNEEDLE. For the purpose of this invention the NEEDLE program from theEMBOSS package was used (version 2.8.0 or higher, EMBOSS: The EuropeanMolecular Biology Open Software Suite (2000) Rice, P. Longden, I. andBleasby, A. Trends in Genetics 16, (6) pp276-277,emboss.bioinformatics.nl/). For protein sequences, EBLOSUM62 is used forthe substitution matrix. For nucleotide sequences, EDNAFULL is used.Other matrices can be specified. The optional parameters used foralignment of amino acid sequences are a gap-open penalty of 10 and a gapextension penalty of 0.5. The skilled person will appreciate that allthese different parameters will yield slightly different results butthat the overall percentage identity of two sequences is notsignificantly altered when using different algorithms.

Global Homology Definition

The homology or identity is the percentage of identical matches betweenthe two full sequences over the total aligned region including any gapsor extensions. The homology or identity between the two alignedsequences is calculated as follows: Number of corresponding positions inthe alignment showing an identical amino acid in both sequences dividedby the total length of the alignment including the gaps. The identitydefined as herein can be obtained from NEEDLE and is labelled in theoutput of the program as “IDENTITY”.

Longest Identity Definition

The homology or identity between the two aligned sequences is calculatedas follows: Number of corresponding positions in the alignment showingan identical amino acid in both sequences divided by the total length ofthe alignment after subtraction of the total number of gaps in thealignment. The identity defined as herein can be obtained from NEEDLE byusing the NOBRIEF option and is labelled in the output of the program as“longest-identity”.

The various embodiments of the invention described herein may becross-combined.

Further embodiments of the invention.

In an embodiment, the yeast cell comprises one or more nucleic acidsequences encoding encoding NAD₊-dependent alcohol dehydrogenaseactivity (EC 1.1.1.1). This enzyme catalyses the conversion ofacetaldehyde into ethanol. The yeast cell may naturally comprise a geneencoding such a dehydrogenase, as is the case with S. cerevisiae(ADH1-5), see ‘Lutstorf and Megnet. 1968 Arch. Biochem. Biophys.126:933-944’, or ‘Ciriacy, 1975, Mutat. Res. 29:315-326’), or a hostcell may be provided with one or more heterologous gene(s) encoding thisactivity, e.g. any or each of the ADH1-5 genes of S. cerevisiae orfunctional homologues thereof may be incorporated into a cell accordingto the invention.

In an embodiment, the yeast cell is selected from Saccharomycetaceae, inparticular from the group of Saccharomyces, such as Saccharomycescerevisiae; Kluyveromyces, such as Kluyveromyces marxianus; Pichia, suchas Pichia stipitis or Pichia angusta; Zygosaccharomyces, such asZygosaccharomyces bailii; and Brettanomyces, such as Brettanomycesintermedius, Issatchenkia, such as Issatchenkia orientalis andHansenula.

In an embodiment, the cell is a prokaryotic cell. In an embodiment thecell is selected from the list consisting of Clostridium, Zymomonas,Thermobacter, Escherichia, Lactobacillus, Geobacillus and Bacillus.

The invention further relates to the use of a yeast cell according tothe invention for the preparation of fermentation product, preferablyethanol. The invention further provides a process for preparingfermentation product, comprising preparing fermentation product fromacetate and from a fermentable carbohydrate—in particular a carbohydrateselected from the group of glucose, fructose, sucrose, maltose, xylose,arabinose, galactose and mannose—which preparation is carried out underanaerobic conditions using a yeast cell according to the invention. Inan embodiment, the preparation is carried out in a fermentation mediumcomprising the acetate and the carbohydrate in a molar ratio is 0.7 orless, in particular at least 0.004 to 0.5, more in particular 0.05 to0.3. In an embodiment of the preparation of fermentation product, atleast part of the carbohydrate and at least part of the acetate has beenobtained by hydrolysing a polysaccharide selected from the group oflignocelluloses, celluloses, hemicelluloses, and pectins. Thelignocellulose is preferably lignocellulosic biomass that has beenhydrolysed thereby obtaining the fermentable carbohydrate and acetate.

In an embodiment, the ligno-cellulosic or hemi-cellulosic material iscontacted with an enzyme composition, wherein one or more sugar isproduced, and wherein the produced sugar is fermented to give afermentation product, wherein the fermentation is conducted with a yeastcell according to the invention.

The fermentation product of the invention may be any useful product. Inone embodiment, it is a product selected from the group consisting ofethanol, n-butanol, isobutanol, lactic acid, 3-hydroxy-propionic acid,acrylic acid, acetic acid, succinic acid, adipic acid, fumaric acid,malic acid, itaconic acid, maleic acid, citric acid, adipic acid, anamino acid, such as lysine, methionine, tryptophan, threonine, andaspartic acid, 1,3-propane-diol, ethylene, glycerol, a β-lactamantibiotic and a cephalosporin, vitamins, pharmaceuticals, animal feedsupplements, specialty chemicals, chemical feedstocks, plastics,solvents, fuels, including biofuels and biogas or organic polymers, andan industrial enzyme, such as a protease, a cellulase, an amylase, aglucanase, a lactase, a lipase, a lyase, an oxidoreductases, atransferase or a xylanase.

In an embodiment, the fermentation product may be one or more ofethanol, butanol, lactic acid, a plastic, an organic acid, a solvent, ananimal feed supplement, a pharmaceutical, a vitamin, an amino acid, anenzyme or a chemical feedstock.

In a preferred embodiment the cell is grown anaerobically. Anaerobicgrowth conditions are herein anaerobic or oxygen limited. Anaerobic ishere defined as a growth process run in the absence of oxygen or inwhich substantially no oxygen is consumed, preferably less than about 5,about 2.5 or about 1 mmol/L/h, and wherein organic molecules serve asboth electron donor and electron acceptors.

An oxygen-limited growth process is a process in which the oxygenconsumption is limited by the oxygen transfer from the gas to theliquid. The degree of oxygen limitation is determined by the amount andcomposition of the ingoing gasflow as well as the actual mixing/masstransfer properties of the fermentation equipment used. Preferably, in aprocess under oxygen-limited conditions, the rate of oxygen consumptionis at least about 5.5, more preferably at least about 6, such as atleast 7 mmol/L/h. A process of the invention comprises recovery of thefermentation product. During fermentation, when acetic acid is present,the ratio of acetic acid/.acetate will depend on pH. The concentrationof acetate in step d) may be chosen similar to the concentration theyeast strain meets in its end use (e.g. in fermentation oflignocellulosic hydrolysate to fermentation product, such hydrolyate maycontain 1-10 g/l acetate, e.g. 2 g/l acetate.

Advantageously, when in accordance with the invention ethanol isproduced, it is produced in a molar ratio of glycerol:ethanol of lessthan 0.04:1, in particular of less than 0.02:1, preferably of less than0.01:1. Glycerol production may be absent (undetectable), although atleast in some embodiments (wherein NADH-dependent glycerol synthesis isreduced yet not completely prohibited) some glycerol may be produced asa side product, e.g. in a ratio glycerol to ethanol of 0.001:1 or more.

The present invention allows complete elimination of glycerolproduction, or at least a significant reduction thereof, by providing arecombinant yeast cell, in particular S. cerevisiae, such that it canreoxidise NADH by the reduction of acetic acid to ethanol viaNADH-dependent reactions.

This is not only advantageous in that glycerol production is avoided orat least reduced, but since the product formed in the re-oxidation ofNADH is also the desired product, namely ethanol, a method of theinvention may also offer an increased product yield (determined as thewt. % of converted feedstock, i.e. carbohydrate plus acetic acid, thatis converted into ethanol). Since acetic acid is generally available atsignificant amounts in lignocellulosic hydrolysates, this makes thepresent invention particularly advantageous for the preparation ofethanol using lignocellulosic biomass as a source for the fermentablecarbohydrate. Further, carbohydrate sources that may contain aconsiderable amount of acetate include sugar beet molasses (hydrolysatesof') and starch containing (e.g. waste products from corn dry millingprocesses, from corn wet milling processes; from starch wastesprocesses, e.g. with stillage recycles).

In a further preferred embodiment, the host cell of the invention has atleast one of: a) the ability of isomerising xylose to xylulose; and, b)the ability to convert L-arabinose into D-xylulose 5-phosphate. For a)the yeast cell preferably has a functional exogenous xylose isomerasegene, which gene confers to the yeast cell the ability to isomerisexylose into xylulose. For b) the yeast cell preferably has functionalexogenous genes coding for a L-arabinose isomerase, a L-ribulokinase anda L-ribulose-5-phosphate 4-epimerase, which genes together confers tothe yeast cell the ability to isomerise convert L-arabinose intoD-xylulose 5-phosphate.

Fungal host cells having the ability of isomerising xylose to xyluloseas e.g. described in WO 03/0624430 and in WO 06/009434. The ability ofisomerising xylose to xylulose is preferably conferred to the yeast cellby transformation with a nucleic acid construct comprising a nucleotidesequence encoding a xylose isomerase. Preferably the yeast cell thusacquires the ability to directly isomerise xylose into xylulose. Morepreferably the yeast cell thus acquires the ability to grow aerobicallyand/or anaerobically on xylose as sole energy and/or carbon sourcethough direct isomerisation of xylose into xylulose (and furthermetabolism of xylulose). It is herein understood that the directisomerisation of xylose into xylulose occurs in a single reactioncatalysed by a xylose isomerase, as opposed to the two step conversionof xylose into xylulose via a xylitol intermediate as catalysed byxylose reductase and xylitol dehydrogenase, respectively.

Several xylose isomerases (and their amino acid and coding nucleotidesequences) that may be successfully used to confer to the yeast cell ofthe invention the ability to directly isomerise xylose into xylulosehave been described in the art. These include the xylose isomerases ofPiromyces sp. and of other anaerobic fungi that belongs to the familiesNeocallimastix, Caecomyces, Piromyces or Ruminomyces (WO 03/0624430),Cyllamyces aberensis (US 20060234364), Orpinomyces (Madhavan et al.,2008, DOI 10.1007/s00253-008-1794-6), the xylose isomerase of thebacterial genus Bacteroides, including e.g. B. thetaiotaomicron (WO06/009434), B. fragilis, and B. uniformis (WO 09/109633), the xyloseisomerase of the anaerobic bacterium Clostridium phytofermentans (Bratet al., 2009, Appl. Environ. Microbiol. 75:2304-2311), and the xyloseisomerases of Clostridium difficile, Ciona intestinales andFusobacterium mortiferum (WO 10/074577).

Fungal host cells having the ability to convert L-arabinose intoD-xylulose 5-phosphate as e.g. described in Wisselink et al. (2007,Appl. Environ. Microbiol. doi:10.1128/AEM.00177-07) and in EP 1 499 708.The ability of to converting L-arabinose into D-xylulose 5-phosphate ispreferably conferred to the yeast cell by transformation with a nucleicacid construct(s) comprising nucleotide sequences encoding a) anarabinose isomerase; b) a ribulokinase, preferably a L-ribulokinase axylose isomerase; and c) a ribulose-5-P-4-epimerase, preferably aL-ribulose-5-P-4-epimerase. Preferably, in the yeast cells of theinvention, the ability to convert L-arabinose into D-xylulose5-phosphate is the ability to convert L-arabinose into D-xylulose5-phosphate through the subsequent reactions of 1) isomerisation ofarabinose into ribulose; 2) phosphorylation of ribulose to ribulose5-phosphate; and, 3) epimerisation of ribulose 5-phosphate intoD-xylulose 5-phosphate. Suitable nucleotide sequences encoding arabinoseisomerases, a ribulokinases and ribulose-5-P-4-epimerases may beobtained from Bacillus subtilis, Escherichia coli (see e.g. EP 1 499708), Lactobacilli, e.g. Escherichia coli (see e.g. Wisselink et al.supra; WO2008/041840), or species of Clavibacter, Arthrobacter andGramella, of which preferably Clavibacter michiganensis, Arthrobacteraurescens and Gramella forsetii (see WO2009/011591).

The transformed cell of the invention further preferably comprisesxylulose kinase activity so that xylulose isomerised from xylose may bemetabolised to pyruvate. Preferably, the yeast cell contains endogenousxylulose kinase activity. More preferably, a cell of the inventioncomprises a genetic modification that increases the specific xylulosekinase activity. Preferably the genetic modification causesoverexpression of a xylulose kinase, e.g. by overexpression of anucleotide sequence encoding a xylulose kinase. The gene encoding thexylulose kinase may be endogenous to the yeast cell or may be a xylulosekinase that is heterologous to the yeast cell. A nucleotide sequencethat may be used for overexpression of xylulose kinase in the yeastcells of the invention is e.g. the xylulose kinase gene from S.cerevisiae (XKS1) as described by Deng and Ho (1990, Appl. Biochem.Biotechnol. 24-25: 193-199). Another preferred xylulose kinase is axylose kinase that is related to the xylulose kinase from Piromyces(xylB; see WO 03/0624430). This Piromyces xylulose kinase is actuallymore related to prokaryotic kinase than to all of the known eukaryotickinases such as the yeast kinase. The eukaryotic xylulose kinases havebeen indicated as non-specific sugar kinases, which have a broadsubstrate range that includes xylulose. In contrast, the prokaryoticxylulose kinases, to which the Piromyces kinase is most closely related,have been indicated to be more specific kinases for xylulose, i.e.having a narrower substrate range. In the yeast cells of the invention,a xylulose kinase to be overexpressed is overexpressed by at least afactor 1.1, 1.2, 1.5, 2, 5, 10 or 20 as compared to a strain which isgenetically identical except for the genetic modification causing theoverexpression. It is to be understood that these levels ofoverexpression may apply to the steady state level of the enzyme'sactivity, the steady state level of the enzyme's protein as well as tothe steady state level of the transcript coding for the enzyme.

A cell of the invention further preferably comprises a geneticmodification that increases the flux of the pentose phosphate pathway asdescribed in WO 06/009434. In an embodiment, the genetic modificationcomprises overexpression of at least one enzyme of the (non-oxidativepart) pentose phosphate pathway. Preferably the enzyme is selected fromthe group consisting of the enzymes encoding for ribulose-5-phosphateisomerase, ribulose-5-phosphate 3-epimerase, transketolase andtransaldolase.

A further preferred cell of the invention comprises a geneticmodification that reduces unspecific aldose reductase activity in theyeast cell. Preferably, unspecific aldose reductase activity is reducedin the host cell by one or more genetic modifications that reduce theexpression of or inactivates a gene encoding an unspecific aldosereductase. Preferably, the genetic modifications reduce or inactivatethe expression of each endogenous copy of a gene encoding an unspecificaldose reductase that is capable of reducing an aldopentose, including,xylose, xylulose and arabinose, in the yeast cell's genome. A given cellmay comprise multiple copies of genes encoding unspecific aldosereductases as a result of di-, poly- or aneuploidy, and/or a cell maycontain several different (iso)enzymes with aldose reductase activitythat differ in amino acid sequence and that are each encoded by adifferent gene. Also in such instances preferably the expression of eachgene that encodes an unspecific aldose reductase is reduced orinactivated. Preferably, the gene is inactivated by deletion of at leastpart of the gene or by disruption of the gene, whereby in this contextthe term gene also includes any non-coding sequence up- or down-streamof the coding sequence, the (partial) deletion or inactivation of whichresults in a reduction of expression of unspecific aldose reductaseactivity in the host cell. A nucleotide sequence encoding an aldosereductase whose activity is to be reduced in the yeast cell of theinvention and amino acid sequences of such aldose reductases aredescribed in WO 06/009434 and include e.g. the (unspecific) aldosereductase genes of S. cerevisiae GRE3 gene (Traff et al., 2001, Appl.Environm. Microbiol. 67: 5668-5674) and orthologues thereof in otherspecies.

In an embodiment, the yeast cell according to the invention may comprisefurther genetic modifications that result in one or more of thecharacteristics selected from the group consisting of (a) increasedtransport of xylose and/or arabinose into the yeast cell; (b) decreasedsensitivity to catabolite repression; (c) increased tolerance toethanol, osmolarity or organic acids; and, (d) reduced production ofby-products. By-products are understood to mean carbon-containingmolecules other than the desired fermentation product and include e.g.xylitol, arabinitol, glycerol and/or acetic acid. Any geneticmodification described herein may be introduced by classical mutagenesisand screening and/or selection for the desired mutant, or simply byscreening and/or selection for the spontaneous mutants with the desiredcharacteristics. Alternatively, the genetic modifications may consist ofoverexpression of endogenous genes and/or the inactivation of endogenousgenes. Genes the overexpression of which is desired for increasedtransport of arabinose and/or xylose into the yeast cell are preferablychosen form genes encoding a hexose or pentose transporter. In S.cerevisiae and other yeasts these genes include HXT1, HXT2, HXT3, HXT4,HXT5, HXT7 and GAL2, of which HXT7, HXT5 and GAL2 are most preferred(see Sedlack and Ho, Yeast 2004; 21: 671-684). Another preferredtransporter for expression in yeast is the glucose transporter encodedby the P. stipitis SUT1 gene (Katahira et al., 2008, Enzyme Microb.Technol. 43: 115-119). Similarly orthologues of these transporter genesin other species may be overexpressed. Other genes that may beoverexpressed in the yeast cells of the invention include genes codingfor glycolytic enzymes and/or ethanologenic enzymes such as alcoholdehydrogenases. Preferred endogenous genes for inactivation includehexose kinase genes e.g. the S. cerevisiae HXK2 gene (see Diderich etal., 2001, Appl. Environ. Microbiol. 67: 1587-1593); the S. cerevisiaeMIG1 or MIG2 genes; genes coding for enzymes involved in glycerolmetabolism such as the S. cerevisiae glycerol-phosphate dehydrogenase 1and/or 2 genes; or (hybridising) orthologues of these genes in otherspecies. Other preferred further modifications of host cells for xylosefermentation are described in van Maris et al. (2006, Antonie vanLeeuwenhoek 90:391-418), WO2006/009434, WO2005/023998, WO2005/111214,and WO2005/091733. Any of the genetic modifications of the yeast cellsof the invention as described herein are, in as far as possible,preferably introduced or modified by self-cloning genetic modification.

A preferred host cell according to the invention has the ability to growon at least one of xylose and arabinose as carbon/energy source,preferably as sole carbon/energy source, and preferably under anaerobicconditions, i.e. conditions as defined herein below for anaerobicfermentation process. Preferably, when grown on xylose as carbon/energysource the host cell produces essentially no xylitol, e.g. the xylitolproduced is below the detection limit or e.g. less than 5, 2, 1, 0.5, or0.3% of the carbon consumed on a molar basis. Preferably, when grown onarabinose as carbon/energy source, the yeast cell produces essentiallyno arabinitol, e.g. the arabinitol produced is below the detection limitor e.g. less than 5, 2, 1, 0.5, or 0.3% of the carbon consumed on amolar basis.

A preferred cell of the invention has the ability to grow on at leastone of a hexose, a pentose, glycerol, acetic acid and combinationsthereof at a rate of at least 0.01, 0.02, 0.05, 0.1, 0.2, 0.25 or 0.3h⁻¹ under aerobic conditions, or, more preferably, at a rate of at least0.005, 0.01, 0.02, 0.05, 0.08, 0.1, 0.12, 0.15 or 0.2 h⁻¹ underanaerobic conditions. Therefore, preferably the host cell has theability to grow on at least one of xylose and arabinose as solecarbon/energy source at a rate of at least 0.01, 0.02, 0.05, 0.1, 0.2,0.25 or 0.3 h⁻¹ under aerobic conditions, or, more preferably, at a rateof at least 0.005, 0.01, 0.02, 0.05, 0.08, 0.1, 0.12, 0.15 or 0.2 h⁻¹under anaerobic conditions. More preferably, the host cell has theability to grow on a mixture of a hexose (e.g. glucose) and at least oneof xylose and arabinose (in a 1:1 weight ratio) as sole carbon/energysource at a rate of at least 0.01, 0.02, 0.05, 0.1, 0.2, 0.25 or 0.3 h⁻¹under aerobic conditions, or, more preferably, at a rate of at least0.005, 0.01, 0.02, 0.05, 0.08, 0.1, 0.12, 0.15 or 0.2 h⁻¹ underanaerobic conditions. Most preferably, the host cell has the ability togrow on a mixture of a hexose (e.g. glucose), at least one of xylose andarabinose and glycerol (in a 1:1:1 weight ratio) as sole carbon/energysource at a rate of at least 0.01, 0.02, 0.05, 0.1, 0.2, 0.25 or 0.3 h⁻¹under aerobic conditions, or, more preferably, at a rate of at least0.005, 0.01, 0.02, 0.05, 0.08, 0.1, 0.12, 0.15 or 0.2 h⁻¹ underanaerobic conditions.

Over the years suggestions have been made for the introduction ofvarious organisms for the production of bio-ethanol from crop sugars. Inpractice, however, all major bio-ethanol production processes havecontinued to use the yeasts of the genus Saccharomyces as ethanolproducer. This is due to the many attractive features of Saccharomycesspecies for industrial processes, i. e., a high acid-, ethanol-andosmo-tolerance, capability of anaerobic growth, and of course its highalcoholic fermentative capacity. Preferred yeast species as host cellsinclude S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum,S. diastaticus, K. lactis, K. marxianus or K. fragilis.

A yeast cell of the invention may be able to convert plant biomass,celluloses, hemicelluloses, pectins, rhamnose, galactose, frucose,maltose, maltodextrines, ribose, ribulose, or starch, starchderivatives, sucrose, lactose and glycerol, for example into fermentablesugars. Accordingly, a cell of the invention may express one or moreenzymes such as a cellulase (an endocellulase or an exocellulase), ahemicellulase (an endo- or exo-xylanase or arabinase) necessary for theconversion of cellulose into glucose monomers and hemicellulose intoxylose and arabinose monomers, a pectinase able to convert pectins intoglucuronic acid and galacturonic acid or an amylase to convert starchinto glucose monomers.

The yeast cell further preferably comprises those enzymatic activitiesrequired for conversion of pyruvate to a desired fermentation product,such as ethanol, butanol, lactic acid, 3-hydroxy-propionic acid, acrylicacid, acetic acid, succinic acid, citric acid, fumaric acid, malic acid,itaconic acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, a13-lactam antibiotic or a cephalosporin.

A preferred cell of the invention is a cell that is naturally capable ofalcoholic fermentation, preferably, anaerobic alcoholic fermentation. Acell of the invention preferably has a high tolerance to ethanol, a hightolerance to low pH (i.e. capable of growth at a pH lower than about 5,about 4, about 3, or about 2.5) and towards organic acids like lacticacid, acetic acid or formic acid and/or sugar degradation products suchas furfural and hydroxy-methylfurfural and/or a high tolerance toelevated temperatures.

Any of the above characteristics or activities of a cell of theinvention may be naturally present in the yeast cell or may beintroduced or modified by genetic modification.

A cell of the invention may be a cell suitable for the production ofethanol. A cell of the invention may, however, be suitable for theproduction of fermentation products other than ethanol. Suchnon-ethanolic fermentation products include in principle any bulk orfine chemical that is producible by a eukaryotic microorganism such as ayeast or a filamentous fungus.

The fermentation process is preferably run at a temperature that isoptimal for the yeast cell. Thus, for most yeasts or fungal host cells,the fermentation process is performed at a temperature which is lessthan about 42° C., preferably less than about 38° C. For yeast orfilamentous fungal host cells, the fermentation process is preferablyperformed at a temperature which is lower than about 35, about 33, about30 or about 28° C. and at a temperature which is higher than about 20,about 22, or about 25° C.

The ethanol yield on xylose and/or glucose in the process preferably isat least about 50, about 60, about 70, about 80, about 90, about 95 orabout 98%. The ethanol yield is herein defined as a percentage of thetheoretical maximum yield.

The invention also relates to a process for producing a fermentationproduct.,

The fermentation processes may be carried out in batch, fed-batch orcontinuous mode. A separate hydrolysis and fermentation (SHF) process ora simultaneous saccharification and fermentation (SSF) process may alsobe applied. A combination of these fermentation process modes may alsobe possible for optimal productivity.

The fermentation process according to the present invention may be rununder aerobic and anaerobic conditions. Preferably, the process iscarried out under micro-aerophilic or oxygen limited conditions.

An anaerobic fermentation process is herein defined as a fermentationprocess run in the absence of oxygen or in which substantially no oxygenis consumed, preferably less than about 5, about 2.5 or about 1mmol/L/h, and wherein organic molecules serve as both electron donor andelectron acceptors.

An oxygen-limited fermentation process is a process in which the oxygenconsumption is limited by the oxygen transfer from the gas to theliquid. The degree of oxygen limitation is determined by the amount andcomposition of the ingoing gasflow as well as the actual mixing/masstransfer properties of the fermentation equipment used. Preferably, in aprocess under oxygen-limited conditions, the rate of oxygen consumptionis at least about 5.5, more preferably at least about 6, such as atleast 7 mmol/L/h. A process of the invention comprises recovery of thefermentation product.

For the recovery of the fermentation product existing technologies areused. For different fermentation products different recovery processesare appropriate. Existing methods of recovering ethanol from aqueousmixtures commonly use fractionation and adsorption techniques. Forexample, a beer still can be used to process a fermented product, whichcontains ethanol in an aqueous mixture, to produce an enrichedethanol-containing mixture that is then subjected to fractionation(e.g., fractional distillation or other like techniques). Next, thefractions containing the highest concentrations of ethanol can be passedthrough an adsorber to remove most, if not all, of the remaining waterfrom the ethanol.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

EXAMPLES

In order to improve the anaerobic (co-)conversion of glycerol and aceticacid both in terms of rate and amount, alternative gene combinationswere tested. For a number of enzymes in the pathway, i.e. glyceroldehydrogenase, dihydroxyacetone kinase and acetaldehyde dehydrogenase,multiple alternative genes were tested that could further enhance theability of the yeast strain to convert glycerol and acetic acid, next topentose and hexose sugars, into ethanol under anaerobic conditions.

The selected enzyme candidates are given in table 8.

TABLE 8 Selected enzyme candidates, encoding desired enzyme activitiesand reference to the protein sequence (SEQ ID NO's are given in thetable) SEQ ID Gene Source organism Enzyme activity NO: adhE Escherichiacoli Bifunctional acetaldehyde- 1 CoA/alcohol dehydrogenase acdHLactobacillus Acetaldehyde dehydrogenase 2 plantarum eutE Escherichiacoli Ethanolamine utilization 3 protein Lin1129 Listeria innocuaAldehyde dehydrogenase 4 adhE Staphylococcus aureus Bifunctionalacetaldehyde- 5 CoA/alcohol dehydrogenase ACS2 Saccharomyces Acetyl-CoAligase 6 cerevisiae gldA Escherichia coli Glycerol dehydrogenase 7 gldAKlebsiella pneumoniae Glycerol dehydrogenase 8 gldA Enterococcusaerogenes Glycerol dehydrogenase 9 gldA Yersinia aldovae Glyceroldehydrogenase 10 DAK1 Saccharomyces Dihydroxyacetone kinase 11cerevisiae dhaK Klebsiella pneumonia Dihydroxyacetone kinase 12 DAK1Yarrowia lipolytica Dihydroxyacetone kinase 13 DAK1 SchizosaccharomycesDihydroxyacetone kinase 14 pombe

The genes were codon-pair-optimized for optimal expression in S.cerevisiae, as described in WO 2008/000632. The SEQ ID NO: of theprotein sequence is indicated in table 8.

Four categories of genes were defined: A) the AADH-group, consisting ofSEQ ID NO:'s 1-5; B) the ACS-group, consisting of SEQ ID NO:6; C) theGLD-group, consisting of SEQ ID NO:'s 7-10 and D) the DAK-group,consisting of SEQ ID NO:'s 11-14.

Expression constructs were made, allowing each gene to be expressed at ahigh level and at a medium/low level.

For group A, the TDH3- and the TDH1-promoters were chosen (SEQ ID NO:'s15 and 16, respectively). The terminator of these genes was thePGK1-terminator (SEQ ID NO: 17), in all cases

For group B, the PGK1- and the PRE3-promoters were chosen (SEQ ID NO:'s18 and 19, respectively). The terminator of these genes was thePGI1-terminator (SEQ ID NO: 20), in all cases

For group C, the ENO1- and the ACT1-promoters were chosen (SEQ ID NO:'s21 and 22, respectively). The terminator of these genes was theCYC1-terminator (SEQ ID NO: 23), in all cases.

For group D, the TPI1- and the ATG7-promoters were chosen (SEQ ID NO:'s24 and 25, respectively). The terminator of these genes was theENO1-terminator (SEQ ID NO: 26), in all cases.

In total, 28 different expression cassettes were assembled, as indicatedin table 9.

TABLE 9 Outline of all assembled expression cassettes (ASS) that weregenerated in a backbone plasmid vector (see Materials and Methods fordetails). The first SEQ ID NO: defines the used promoter, the second SEQID NO: defines the ORF and the third SEQ ID NO: defines thetranscription terminator. The ASS-number is generated for easierreference. Group A SEQ ID NO: 15 - SEQ ID NO: 1 - SEQ ID NO: 17 ASS01SEQ ID NO: 15 - SEQ ID NO: 2 - SEQ ID NO: 17 ASS02 SEQ ID NO: 15 - SEQID NO: 3 - SEQ ID NO: 17 ASS03 SEQ ID NO: 15 - SEQ ID NO: 4 - SEQ ID NO:17 ASS04 SEQ ID NO: 15 - SEQ ID NO: 5 - SEQ ID NO: 17 ASS05 SEQ ID NO:16 - SEQ ID NO: 1 - SEQ ID NO: 17 ASS06 SEQ ID NO: 16 - SEQ ID NO: 2 -SEQ ID NO: 17 ASS07 SEQ ID NO: 16 - SEQ ID NO: 3 - SEQ ID NO: 17 ASS08SEQ ID NO: 16 - SEQ ID NO: 4 - SEQ ID NO: 17 ASS09 SEQ ID NO: 16 - SEQID NO: 5 - SEQ ID NO: 17 ASS10 Group B SEQ ID NO: 18 - SEQ ID NO: 6 -SEQ ID NO: 20 ASS11 SEQ ID NO: 19 - SEQ ID NO: 6 - SEQ ID NO: 20 ASS12Group C SEQ ID NO: 21 - SEQ ID NO: 7 - SEQ ID NO: 23 ASS13 SEQ ID NO:21 - SEQ ID NO: 8 - SEQ ID NO: 23 ASS14 SEQ ID NO: 21 - SEQ ID NO: 9 -SEQ ID NO: 23 ASS15 SEQ ID NO: 21 - SEQ ID NO: 10 - SEQ ID NO: 23 ASS16SEQ ID NO: 22 - SEQ ID NO: 7 - SEQ ID NO: 23 ASS17 SEQ ID NO: 22 - SEQID NO: 8 - SEQ ID NO: 23 ASS18 SEQ ID NO: 22 - SEQ ID NO: 9 - SEQ ID NO:23 ASS19 SEQ ID NO: 22 - SEQ ID NO: 10 - SEQ ID NO: 23 ASS20 Group D SEQID NO: 24 - SEQ ID NO: 11 - SEQ ID NO: 26 ASS21 SEQ ID NO: 24 - SEQ IDNO: 12 - SEQ ID NO: 26 ASS22 SEQ ID NO: 24 - SEQ ID NO: 13 - SEQ ID NO:26 ASS23 SEQ ID NO: 24 - SEQ ID NO: 14 - SEQ ID NO: 26 ASS24 SEQ ID NO:25 - SEQ ID NO: 11 - SEQ ID NO: 26 ASS25 SEQ ID NO: 25 - SEQ ID NO: 12 -SEQ ID NO: 26 ASS26 SEQ ID NO: 25 - SEQ ID NO: 13 - SEQ ID NO: 26 ASS27SEQ ID NO: 25 - SEQ ID NO: 14 - SEQ ID NO: 26 ASS28

The assembled expression cassettes (ASS) were amplified by PCR usingprimers with connectors at each flank, as depicted in FIG. 2. ThePCR-product is designated a CAS. The CAS elements overlap partly witheither an integration flank and/or another CAS and/or a selectablemarker, allowing upon introduction in competent yeast cellsrecombination of the genetic elements into the genome of thetransformation competent yeast strain, as depicted in FIG. 2.

In a multifactorial pathway design, all possible combinations of theindividual members of the four categories (group A, B, C and D) werecombined, using the technology described in patent applicationPCT/EP2013/056623. All 1280 different possible combinations ofexpression cassettes were generated and used to transform strain RN1069,a gpd1gpd2 double deletion strain (see Material and Methods).

As depicted in FIG. 2, an antibiotic resistance marker was included inthe multifactorial pathway design, in order to enable selection of yeasttransformants that had successfully integrated the desired pathway intotheir genome by recombination. The sequence of the selection marker isgiven as SEQ ID NO: 27.

In addition, two integration flanks, designated 5′-INT1 and 3′-INT1 inFIG. 2, were included in the transformation mixture. The sequences ofthese two flanking regions are generated in a PCR reaction using genomicDNA of the host strain as template and primer combination SEQ ID NO: 30and 31 for the 5′-flank of INT1(figure 2) and primer combination SEQ IDNO: 42 and 43 for the 3′-INT1 flank (FIG. 2).

Materials and Methods

General Molecular Biology Techniques

Unless indicated otherwise, the methods used are standard biochemicaltechniques. Examples of suitable general methodology textbooks includeSambrook et al., Molecular Cloning, a Laboratory Manual (1989) andAusubel et al., Current Protocols in Molecular Biology (1995), JohnWiley & Sons, Inc.

Media

The media used in the experiments was either YEP-medium (10 g/l yeastextract, 20 g/l peptone) or solid YNB-medium (6.7 g/l yeast nitrogenbase, 15 g/l agar), supplemented with sugars as indicated in theexamples. For solid YEP medium, 15 g/l agar was added to the liquidmedium prior to sterilization.

In the anaerobic screening experiment, Mineral Medium was used. Thecomposition of Mineral Medium has been described by Verduyn et al.(Yeast (1992), Volume 8, 501-517). The use of ammoniumsulfate washowever omitted; instead, as a nitrogen source, 2.3 g/l urea was used.In addition, ergosterol (0.01 g/L), Tween80 (0.42 g/L) and sugars (asindicated) were added.

Transformants of strain RN1069 (by integration of DNA constructs) arehistidine auxotrophic.

Strains

The strains used in the experiments were RN1041 and RN1069.RN1041 hasbeen described in WO 2012/067510. This strain has the followinggenotype:

MAT a, ura3-52, leu2-112, his3::loxP, gre3::loxP, loxP-pTPI1::TAL1,loxP-pTPI1::RKI1, loxP-pTPI1-TKL1, loxP-pTPI1-RPE1,delta::pADH1-XKS1-tCYC1-LEU2, delta:: URA3-pTPI1-xylA-tCYC1

MAT a=mating type a

ura3-52, leu2-112, HIS3::loxP mutations in the URA3, LEU2 and HIS3 genesrespectively. The ura3-52 mutation is complemented by the URA3 gene onthe Piromyces xylA overexpression construct; the leu2-112 mutation iscomplemented by the LEU2 gene on the XKS1 overexpression construct. Thedeletion of the H/S3-gene causes a histidine auxotrophy. For thisreason, RN1041 needs histidine in the medium for growth.

gre3::loxP is a deletion of the GRE3 gene, encoding aldose reductase.The loxP site is left behind in the genome after marker removal.

loxP-pTPI1 designates the overexpression of genes of, in the experimentsdescribed herein, the non-oxidative pentose phosphate pathway byreplacement of the native promoter by the promoter of the TPI1 gene. TheloxP site upstream of the strong, constitutive TPI1 promoter remains inthe genome after marker removal (Kuyper et al, FEMS Yeast Research 5(2005) 925-934).

delta:: means chromosomal integration of the construct afterrecombination on the long terminal repeats of the Ty1 retrotransposon.

Strain RN1001 is the parent strain of strain RN1041, i.e. beforedeletion of the HIS3-gene.

Strain RN1069 is derived from RN1041: the GPD1 and GPD2 genes weredisrupted by gene replacement. To this end, dominant antibioticresistance markers, flanked by sequences homologous to the sequencesjust beside the open reading frame (ORF) of GPD1 or GPD2, wereconstructed by PCR and used to transform strain RN1041. These genedisruption cassettes have been included in the sequence listing as SEQID NO: 28 and 29 respectively. The construction of strain RN1069 is alsodescribed in detail in WO2013/081456. The genotype of strain RN1069 is:

MAT a, ura3-52, leu2-112, his3::loxP, gre3::loxP, loxP-pTPI1::TAL1,loxP-pTPI1::RKI1, loxP-pTPI1-TKL1, loxP-pTPI1-RPE1,delta::pADH1-XKS1-tCYC1-LEU2, delta::URA3-pTPI1-xylA-tCYC1 gpd1::hphMX,gpd2::natMX.

Strain RN1189 was used as a reference strain. Strain RN1189 is describedin WO2013/081456. In short, strain RN1189 was constructed bytransformation of strain RN1069 with plasmid pRN977. Plasmid pRN977 is a2μ plasmid and contains the following features: the H/S3-gene forselection of transformants, the ampicillin resistance marker forselection in E. coli, the adhE-gene from E. coli under control of thePGK1-promoter and the ADH1-terminator, the DAK1-gene from S. cerevisiaeunder control of the TPI1-promoter and the PGI1-terminator and the E.coli gldA-gene, under control of the ACT1-promoter and CYC1-terminator.All promoters and terminators are from S. cerevisiae.

Strain Construction

The strain construction approach is described in patent applicationPCT/EP2013/056623. It describes the techniques enabling the constructionof expression cassettes from various genes of interest in such a way,that these cassettes are combined into a pathway and integrated in aspecific locus of the yeast genome upon transformation of this yeast. Aschematic representation is depicted in FIG. 2.

Firstly, an integration site in the yeast genome is chosen (e.g. INT1).A DNA fragment of approximately 500 bp of the up- and downstream part ofthe integration locus is amplified using PCR, flanked by a connector.These connectors are 50 bp sequences that allow for correct in vivorecombination of the pathway upon transformation in yeast (Saccharomycescerevisiae e.g.). The genes of interest, as well as a selectableresistance marker (e.g. kanMX), are generated by PCR, incorporating adifferent connector at each flank, as is displayed in FIG. 2. Upontransformation of yeast cells with the DNA fragments, in vivorecombination and integration into the genome takes place at the desiredlocation. This technique allows for pathway tuning, as one or more genesfrom the pathway can be replaced with (an)other gene(s) or geneticelement(s), as long as that the connectors that allow for homologousrecombination remain constant (patent application PCT/EP2013/056623).

Expression Cassette Construction

The open reading frames (ORFs), promoter sequences and terminators weresynthesized at DNA 2.0 (Menlo Park, Calif. 94025, USA). The sequences ofthese genetic elements are listed as SEQ ID NO:'s 1 until and including26. The promoter, ORF and terminator sequences were assembled by usingthe Golden Gate technology, as described by Engler et al (2011) andreferences therein. The assembled expression cassettes were ligated intoBsal-digested backbone vectors; group A (table 2) into p5Abbn (SEQ IDNO: 44), group B (table 2) into pBCbbn (SEQ ID NO: 45), group C (table2) into pCDbbn (SEQ ID NO: 46) and group D (table 2) into pD3bbn (SEQ IDNO: 47).

Expression Cassette Amplification

The assembled expression cassettes that were generated as describedabove (section “Expression cassette construction”), the integrationflanks and the selective marker were amplified by PCR using the primersdescribed as SEQ ID NO:'s 30-43 (see below). The kanMX-marker, as G418resistance marker for selection in yeast, was amplified from a plasmidcontaining this marker. The sequence of the marker is described as SEQID NO: 27.

The INT1-flanks were amplified from genomic DNA from strain CEN.PK113-7Dusing the SEQ ID NO:'s 30 and 31 (5′-INT1 flank) and 42 and 43 (3′-INT1flank).

TABLE 10 Overview of primers use to amplify CAS (expression cassetteswith overlapping sequences for homologous recombination in yeast) fromASS (assembled expression cassettes). The corresponding number are used(e.g. CAS01 is from ASS01). Forward Reverse CAS Element primer primer —5′-INT1 SEQ ID NO: 30 SEQ ID NO: 31 (SEQ ID NO: 28) CAS01- AADH inp5Abbn SEQ ID NO: 32 SEQ ID NO: 33 CAS10 — marker SEQ ID NO: 34 SEQ IDNO: 35 (SEQ ID NO: 27) CAS11- ACS in pBCbbn SEQ ID NO: 36 SEQ ID NO: 37CAS12 CAS13- GLDA in pCDbbn SEQ ID NO: 38 SEQ ID NO: 39 CAS20 CAS21- DAKin pD3bbn SEQ ID NO: 40 SEQ ID NO: 41 CAS28 — 3′-INT1 SEQ ID NO: 42 SEQID NO: 43 (SEQ ID NO: 29)

Transformation of Yeast Cells

Yeast transformation was done according to the method described bySchiestl and Gietz (Current Genetics (1989), Volume 16, 339-346).

Anaerobic Growth Experiments in Microplates

Growth experiments were performed in flat bottom NUNC microplates(MTPs). 275 μl of medium was filled out in each well. The composition ofthe medium was as follows: Mineral medium (based on Verduyn et al(1992), urea instead of ammoniumsulfate)

-   2% glucose-   2% xylose-   1% glycerol-   2 g/l acetic acid-   200 μg/ml histidine (in case of strain RN1069 and derivatives, which    are his3::loxP) pH 4.5

All MTPs were sealed with an aluminum seal. The MTPs were then placed inthe anaerobic incubator (Infors). After 48 hours of growth, the MTPswere removed from the anaerobic Infors. The plates were then spun down10 minutes @ 2750 rpm in a microplate centrifuge. 150 μl of thesupernatant was then transferred to a MTP suitable for NMR analysis.

NMR Analysis

For the quantification of glucose, xylose, glycerol, acetic acid andethanol in the sample, 150 μl sample is transferred accurately into asuitable vial. Subsequently 100 μl internal standard solution,containing maleic acid (20 g/l), EDTA (40 g/l) and trace amounts of DSS(4,4-dimethyl-4-silapentane-1-sulfonic acid) in D₂O, and 450 A D₂O isadded.

1D ¹H NMR spectra are recorded on a Bruker Avance III 700 MHz, equippedwith a cryo-probe, using a pulse program with water suppression (powercorresponding to 3 Hz) at a temperature of 27° C.

The analyte concentrations are calculated based on the following signals(δ relative to DSS):

α-glucose peak at 5.22 ppm (d, 0.38H, J=4 Hz),

α-xylose peak at 5.18 ppm (d, 0.37H, J=4 Hz),

glycerol peak at 3.55 ppm (dd, 2H, J_(1,2)=6 Hz and J_(1a,1b)=12 Hz)

acetic acid peak at 1.91 ppm (s, 3H)

ethanol peak at 1.17 ppm (t, 3H, J=7 Hz)

The signal user for the standard:

Maleic acid peak at 6.05 ppm (s, 2H)

Example 1 Construction of the Full Combinatorial Array of YeastTransformants

The full combinatorial array of strains was constructed as describedabove. To this end, strain RN1069 was transformed with all 1280 mixes ofgenes (vide supra). Transformation mixes were plated on YEP-agarcontaining 20 g glucose per liter and 200 μg G418/ml. From eachtransformation, two independent transformants were selected andtransferred to YEPD agar in microplates (one colony per well). On eachmicroplate, a reference strains was inoculated as well: RN1189.

Example 2 Growth Experiment and Analysis of the Results

The array of selected colonies and strains (Example 1) was tested in theexperimental set-up described in the Material and Methods.

In short, the strains in the microplate with YEPD-agar were used toinoculate, 275 μl Mineral Medium containing 200 μg histidine per ml, 2%glucose, 2% xylose, 1% glycerol and 2 g/l acetic acid, in microplates.The pH of the medium was set at 4.5, below the pKa of acetic acid. Themicroplate was sealed and incubated for 48 hours under anaerobicconditions. Cells were spun down by centrifugation and the supernatantwas analyzed by NMR. The top 150 results are given in table 11.

TABLE 11 Results of the growth experiments of the 150 best strains (top150). In the first two columns the identification of the strainexperiments is given. In columns 3-6, the construct of the strain isgiven, for AADH, ACS, GLDA and DAK1. Columns 7-11 indicates the resultsof the fermentation, concentrations (g/l) of glucose, xylose, glycerol,acetate and ethanol, respectively. Columns 12-14 give the ranking ofstrains for each product (glycerol and acetate consumption) and ethanolproduction. Column 15 gives total ranking based on the rankings incolumn 12-14. Glycerol Acetate EtOH Total Num Expno AADH ACS GLDA DAK1glucose xylose glycerol acetate ethanol Rank Rank Rank Rank 355 67 CAS2CAS11 CAS13 CAS21 0.0 0.8 7.0 0.3 18.9 2 2 11 1 1131 75 CAS5 CAS11 CAS15CAS24 0.1 2.1 7.6 0.5 18.4 27 12 20 2 820 52 CAS3 CAS12 CAS18 CAS23 0.11.1 7.8 0.4 18.2 41 3 17 3 386 2 CAS2 CAS11 CAS15 CAS21 0.1 2.2 7.3 0.417.2 5 4 54 4 354 66 CAS2 CAS11 CAS13 CAS21 0.1 1.1 7.6 0.6 18.5 23 3014 5 1696 64 CAS7 CAS12 CAS15 CAS23 0.1 2.0 7.6 0.6 18.6 22 28 18 6 78719 CAS3 CAS12 CAS16 CAS23 0.1 0.5 7.8 0.5 18.3 51 7 13 7 1130 74 CAS5CAS11 CAS15 CAS24 0.1 3.8 7.4 0.7 21.5 10 54 8 8 519 39 CAS2 CAS12 CAS15CAS23 0.1 2.9 7.3 0.6 17.8 4 32 42 9 2571 75 CAS2 CAS11 CAS19 CAS25 0.22.0 7.6 0.6 17.4 24 26 33 10 520 40 CAS2 CAS12 CAS15 CAS23 0.1 1.5 7.60.6 18.0 26 38 21 11 608 32 CAS3 CAS11 CAS13 CAS21 0.1 2.4 7.4 0.4 16.97 6 73 12 522 42 CAS2 CAS12 CAS15 CAS24 0.1 1.1 7.6 0.6 17.7 21 43 23 131682 50 CAS7 CAS12 CAS14 CAS23 0.1 2.6 7.4 0.7 18.2 11 50 28 14 1697 65CAS7 CAS12 CAS15 CAS24 0.1 1.6 7.7 0.6 18.6 30 44 15 14 359 71 CAS2CAS11 CAS13 CAS23 0.1 2.9 7.1 0.5 16.7 3 10 86 16 1098 42 CAS5 CAS11CAS13 CAS21 0.1 1.4 7.5 0.5 16.3 15 9 77 17 632 56 CAS3 CAS11 CAS14CAS24 0.1 2.0 7.4 0.6 16.8 12 23 68 18 1119 63 CAS5 CAS11 CAS14 CAS240.1 3.1 7.8 0.6 17.8 46 19 46 19 614 38 CAS3 CAS11 CAS13 CAS24 0.1 1.37.9 0.6 17.6 57 31 27 20 506 26 CAS2 CAS12 CAS14 CAS24 0.1 2.3 7.6 0.617.4 28 45 50 21 739 67 CAS3 CAS12 CAS13 CAS23 0.1 2.4 7.7 0.6 16.8 3520 74 22 1677 45 CAS7 CAS12 CAS14 CAS21 0.1 1.4 7.8 0.7 17.3 47 55 31 231539 3 CAS7 CAS11 CAS13 CAS23 0.1 4.4 7.5 0.6 17.4 14 27 96 24 784 16CAS3 CAS12 CAS16 CAS21 0.1 1.6 8.1 0.6 18.2 100 21 19 25 1695 63 CAS7CAS12 CAS15 CAS23 0.1 4.4 7.6 0.6 17.2 18 37 103 26 2263 55 CAS9 CAS11CAS14 CAS24 0.1 1.6 7.7 0.6 16.0 36 29 94 27 2452 52 CAS10 CAS12 CAS15CAS21 0.1 3.8 7.5 0.6 16.6 17 22 134 28 1043 83 CAS4 CAS12 CAS16 CAS210.1 1.3 8.0 0.7 17.6 88 64 26 29 630 54 CAS3 CAS11 CAS14 CAS23 0.0 1.68.0 0.7 17.3 79 63 37 30 1707 75 CAS7 CAS12 CAS16 CAS21 0.1 3.8 7.8 0.717.9 53 73 57 31 641 65 CAS3 CAS11 CAS15 CAS21 0.0 2.1 7.9 0.7 17.4 7170 47 32 615 39 CAS3 CAS11 CAS13 CAS24 0.1 3.2 7.9 0.7 16.7 56 60 95 33214 22 CAS1 CAS12 CAS13 CAS23 0.2 4.9 6.3 0.3 16.5 1 1 211 34 1233 81CAS5 CAS12 CAS15 CAS21 0.1 4.1 7.4 0.5 16.2 8 13 198 35 358 70 CAS2CAS11 CAS13 CAS23 0.1 2.6 7.9 0.8 17.1 62 98 67 36 1252 4 CAS5 CAS12CAS16 CAS23 0.1 4.2 8.0 0.6 17.3 89 48 93 37 488 8 CAS2 CAS12 CAS13CAS23 0.1 2.3 7.8 0.6 15.6 40 40 152 38 1105 49 CAS5 CAS11 CAS13 CAS240.1 2.2 8.2 0.8 26.0 139 93 1 39 1540 4 CAS7 CAS11 CAS13 CAS23 0.1 3.57.4 0.5 15.8 13 15 208 40 2564 68 CAS2 CAS11 CAS19 CAS21 0.1 2.2 7.9 0.615.9 65 46 126 41 783 15 CAS3 CAS12 CAS16 CAS21 0.2 2.6 8.3 0.7 17.6 14558 36 42 536 56 CAS2 CAS12 CAS16 CAS24 0.0 2.0 8.1 0.8 17.3 105 96 48 432555 59 CAS2 CAS11 CAS18 CAS25 0.0 1.7 7.6 0.6 14.9 19 17 215 44 500 20CAS2 CAS12 CAS14 CAS21 0.1 2.1 8.0 0.7 16.3 85 82 89 45 2187 75 CAS9CAS12 CAS14 CAS23 0.1 2.9 8.0 0.8 17.0 77 104 76 46 626 50 CAS3 CAS11CAS14 CAS21 0.1 1.9 8.2 0.7 17.0 120 85 53 47 837 69 CAS3 CAS12 CAS19CAS23 0.0 0.9 8.4 0.7 18.2 184 62 16 48 535 55 CAS2 CAS12 CAS16 CAS240.1 2.7 8.1 0.8 17.6 102 124 43 49 774 6 CAS3 CAS12 CAS15 CAS24 0.2 4.57.9 0.6 16.4 61 18 194 50 736 64 CAS3 CAS12 CAS13 CAS21 0.2 4.0 7.9 0.716.6 66 78 135 51 2168 56 CAS9 CAS12 CAS13 CAS21 0.1 1.9 7.9 0.8 15.9 7099 111 52 1207 55 CAS5 CAS12 CAS13 CAS24 0.0 2.2 7.5 0.5 14.8 16 11 26553 503 23 CAS2 CAS12 CAS14 CAS23 0.1 2.3 7.9 0.7 15.7 74 75 148 54 255256 CAS2 CAS11 CAS18 CAS23 0.1 3.8 7.4 0.6 15.6 6 24 269 55 839 71 CAS3CAS12 CAS19 CAS24 0.2 3.9 8.0 0.7 16.5 87 71 144 56 521 41 CAS2 CAS12CAS15 CAS24 0.1 3.8 8.1 0.8 17.0 99 109 98 57 740 68 CAS3 CAS12 CAS13CAS23 0.1 4.0 7.9 0.7 16.3 76 59 176 58 2086 70 CAS9 CAS11 CAS15 CAS240.1 3.1 7.7 0.7 15.7 38 84 192 59 2565 69 CAS2 CAS11 CAS19 CAS22 0.1 4.57.9 0.6 16.2 58 41 225 60 2276 68 CAS9 CAS11 CAS15 CAS23 0.1 2.1 8.2 0.715.7 115 81 136 61 406 22 CAS2 CAS11 CAS16 CAS23 0.1 1.8 8.2 0.7 15.6137 67 132 62 758 86 CAS3 CAS12 CAS14 CAS24 0.0 2.2 7.8 0.5 14.6 44 8298 63 2440 40 CAS10 CAS12 CAS14 CAS23 0.1 3.5 8.0 0.7 16.0 86 90 174 632553 57 CAS2 CAS11 CAS18 CAS24 0.2 5.3 7.7 0.6 15.8 29 16 328 65 790 22CAS3 CAS12 CAS16 CAS24 0.1 2.9 8.5 0.7 17.3 230 79 66 66 631 55 CAS3CAS11 CAS14 CAS24 0.2 4.2 8.1 0.7 16.1 101 77 201 67 407 23 CAS2 CAS11CAS16 CAS23 0.0 1.4 8.0 0.7 14.5 78 61 244 68 751 79 CAS3 CAS12 CAS14CAS21 0.1 3.5 8.3 0.8 16.3 147 92 149 69 2549 53 CAS2 CAS11 CAS18 CAS220.2 4.7 7.4 0.6 15.5 9 39 341 70 499 19 CAS2 CAS12 CAS14 CAS21 0.1 3.38.1 0.8 16.2 111 135 156 71 655 79 CAS3 CAS11 CAS15 CAS28 0.1 1.4 8.70.7 16.8 286 66 55 72 1303 55 CAS5 CAS12 CAS19 CAS24 0.1 5.3 8.0 0.716.4 84 72 261 73 788 20 CAS3 CAS12 CAS16 CAS23 0.1 5.5 7.8 0.4 15.9 505 363 74 1138 82 CAS5 CAS11 CAS16 CAS21 0.1 2.5 8.2 0.9 16.0 130 159 12974 2056 40 CAS9 CAS11 CAS13 CAS24 0.1 3.3 7.9 0.8 15.6 73 123 229 762069 53 CAS9 CAS11 CAS14 CAS23 0.0 3.6 7.9 0.8 15.6 63 120 249 77 735 63CAS3 CAS12 CAS13 CAS21 0.2 4.3 8.3 0.8 16.6 154 128 154 78 1458 18 CAS6CAS12 CAS15 CAS21 0.1 4.6 8.1 0.7 16.0 96 76 268 79 815 47 CAS3 CAS12CAS17 CAS28 0.1 2.1 8.9 0.7 17.6 344 68 32 80 1216 64 CAS5 CAS12 CAS14CAS21 0.1 4.6 8.1 0.8 16.1 108 102 236 81 1276 28 CAS5 CAS12 CAS17 CAS270.1 2.3 8.4 1.0 17.0 188 218 65 82 629 53 CAS3 CAS11 CAS14 CAS23 0.1 4.28.3 0.8 16.1 142 118 218 83 836 68 CAS3 CAS12 CAS19 CAS23 0.1 2.0 8.80.8 17.4 303 137 38 83 642 66 CAS3 CAS11 CAS15 CAS21 0.2 5.0 8.0 0.816.0 92 107 282 85 1553 17 CAS7 CAS11 CAS14 CAS23 0.1 3.1 7.6 0.7 14.425 49 413 86 998 38 CAS4 CAS12 CAS13 CAS23 0.1 2.8 8.5 1.0 17.3 201 24562 87 1031 71 CAS4 CAS12 CAS15 CAS23 0.1 3.6 8.3 0.9 16.2 161 189 160 88613 37 CAS3 CAS11 CAS13 CAS23 0.1 3.0 8.3 0.7 15.1 169 65 280 89 821 53CAS3 CAS12 CAS18 CAS23 0.1 2.0 8.8 0.9 17.2 326 141 49 90 1016 56 CAS4CAS12 CAS14 CAS23 0.1 2.6 8.4 1.0 16.6 175 261 87 91 1713 81 CAS7 CAS12CAS16 CAS24 0.1 3.9 7.9 0.7 14.9 68 74 387 92 755 83 CAS3 CAS12 CAS14CAS23 0.1 4.1 7.7 0.6 14.5 31 25 486 93 1666 34 CAS7 CAS12 CAS13 CAS240.1 3.3 8.1 0.8 15.0 107 113 324 94 1001 41 CAS4 CAS12 CAS13 CAS24 0.14.7 8.3 0.9 16.4 163 178 207 95 1268 20 CAS5 CAS12 CAS17 CAS23 0.1 3.38.7 0.9 16.7 270 180 99 96 1112 56 CAS5 CAS11 CAS14 CAS21 0.1 3.3 8.30.8 15.2 164 110 277 97 1206 54 CAS5 CAS12 CAS13 CAS24 0.2 5.2 7.7 0.715.1 37 56 463 98 1012 52 CAS4 CAS12 CAS14 CAS21 0.1 2.8 8.5 1.0 16.9213 268 81 99 752 80 CAS3 CAS12 CAS14 CAS21 0.1 4.7 8.3 0.9 16.1 156 145264 100 1107 51 CAS5 CAS11 CAS13 CAS25 0.1 2.0 8.7 1.0 17.3 277 243 45100 791 23 CAS3 CAS12 CAS16 CAS25 0.1 2.1 9.0 0.8 17.2 384 133 52 102903 39 CAS4 CAS11 CAS15 CAS24 0.1 3.2 8.6 1.0 16.7 241 236 97 103 128032 CAS5 CAS12 CAS18 CAS21 0.1 2.9 8.4 1.0 15.9 185 235 157 104 1805 77CAS8 CAS11 CAS14 CAS23 0.2 5.5 7.9 0.8 15.7 72 126 385 105 1027 67 CAS4CAS12 CAS15 CAS21 0.1 3.6 8.4 1.0 16.2 193 229 163 106 1664 32 CAS7CAS12 CAS13 CAS23 0.2 4.5 8.3 0.9 15.8 149 151 285 106 483 3 CAS2 CAS12CAS13 CAS21 0.1 5.0 8.5 0.8 16.4 227 125 239 108 2188 76 CAS9 CAS12CAS14 CAS23 0.1 3.8 8.1 0.9 15.0 94 164 343 109 1034 74 CAS4 CAS12 CAS15CAS24 0.1 3.0 8.7 1.0 17.2 268 264 70 110 612 36 CAS3 CAS11 CAS13 CAS230.1 4.0 7.6 0.5 13.9 20 14 573 111 789 21 CAS3 CAS12 CAS16 CAS24 0.2 6.78.1 0.6 15.8 104 33 475 112 1220 68 CAS5 CAS12 CAS14 CAS23 0.2 4.2 7.70.6 14.2 32 42 540 113 1269 21 CAS5 CAS12 CAS17 CAS23 0.1 3.6 8.6 0.916.0 257 174 185 114 1106 50 CAS5 CAS11 CAS13 CAS25 0.0 1.3 9.0 1.0 22.3397 216 6 115 1047 87 CAS4 CAS12 CAS16 CAS23 0.1 3.5 8.3 0.8 14.8 141122 366 116 490 10 CAS2 CAS12 CAS13 CAS24 0.1 3.7 7.7 0.6 13.9 34 47 552117 1711 79 CAS7 CAS12 CAS16 CAS23 0.2 5.6 8.2 0.9 15.8 126 149 358 117534 54 CAS2 CAS12 CAS16 CAS23 0.1 5.3 8.4 0.9 16.3 173 195 273 119 155519 CAS7 CAS11 CAS14 CAS24 0.1 3.4 8.2 0.8 14.5 117 105 426 120 1554 18CAS7 CAS11 CAS14 CAS23 0.1 4.5 7.8 0.8 14.6 52 95 504 121 1044 84 CAS4CAS12 CAS16 CAS21 0.1 2.9 8.8 0.9 16.0 318 192 143 122 887 23 CAS4 CAS11CAS14 CAS24 0.0 2.0 8.8 1.0 15.8 314 213 127 123 1699 67 CAS7 CAS12CAS15 CAS25 0.1 2.0 8.9 1.0 17.5 348 272 34 123 616 40 CAS3 CAS11 CAS13CAS25 0.0 1.5 9.0 1.0 17.1 391 225 40 125 1683 51 CAS7 CAS12 CAS14 CAS240.3 6.8 8.4 0.9 16.6 181 169 315 126 1329 81 CAS6 CAS11 CAS13 CAS21 0.25.0 8.2 0.8 15.1 124 111 431 127 2550 54 CAS2 CAS11 CAS18 CAS22 0.1 2.78.8 1.0 16.3 315 246 113 128 1324 76 CAS5 CAS12 CAS20 CAS27 0.2 3.6 8.71.0 15.9 264 228 191 129 1565 29 CAS7 CAS11 CAS15 CAS21 0.2 4.7 8.3 0.915.4 152 170 361 129 2523 27 CAS2 CAS11 CAS16 CAS25 0.2 4.5 8.4 0.9 15.7195 200 288 129 656 80 CAS3 CAS11 CAS15 CAS28 0.1 2.3 9.0 0.9 16.2 414166 104 132 391 7 CAS2 CAS11 CAS15 CAS23 0.1 4.3 8.4 0.8 15.1 172 136381 133 796 28 CAS3 CAS12 CAS16 CAS27 0.1 2.3 9.0 1.0 16.9 388 232 71134 322 34 CAS1 CAS12 CAS19 CAS21 0.2 4.5 8.6 1.0 15.9 238 212 246 135628 52 CAS3 CAS11 CAS14 CAS22 0.1 2.0 9.1 0.9 16.8 444 196 64 136 507 27CAS2 CAS12 CAS14 CAS25 0.1 1.8 8.8 1.1 16.8 316 328 61 137 1302 54 CAS5CAS12 CAS19 CAS24 0.1 6.3 8.1 0.8 15.6 106 116 484 138 822 54 CAS3 CAS12CAS18 CAS24 0.0 0.8 9.1 1.0 17.5 437 248 25 139 545 65 CAS2 CAS12 CAS17CAS21 0.1 1.5 8.8 1.1 17.5 323 358 30 140 920 56 CAS4 CAS11 CAS16 CAS240.1 3.2 8.7 1.0 16.0 292 260 162 141 795 27 CAS3 CAS12 CAS16 CAS27 0.11.9 9.2 0.9 17.1 520 146 51 142 882 18 CAS4 CAS11 CAS14 CAS21 0.1 2.58.9 1.0 16.2 341 265 115 143 746 74 CAS3 CAS12 CAS13 CAS26 0.1 2.9 8.71.0 15.5 266 258 214 144 832 64 CAS3 CAS12 CAS19 CAS21 0.2 5.4 8.5 0.915.6 209 157 372 144 1018 58 CAS4 CAS12 CAS14 CAS24 0.1 3.5 8.7 1.0 16.3287 292 159 144 368 80 CAS2 CAS11 CAS13 CAS28 0.1 0.9 8.9 1.1 17.5 374343 24 147 581 5 CAS2 CAS12 CAS19 CAS23 0.0 2.5 8.0 0.7 13.1 82 51 611148 423 39 CAS2 CAS11 CAS17 CAS23 0.1 3.7 8.6 1.0 15.6 251 237 259 149756 84 CAS3 CAS12 CAS14 CAS23 0.2 6.2 7.7 0.7 14.7 39 83 628 150

In FIG. 3, the residual acetic acid concentration in the fermentationbroth is plotted as a function of the residual glycerol concentration.The results clearly show that there is a strong correlation between theresidual glycerol concentration and the residual acetic acidconcentration: the lower the residual glycerol concentration afterfermentation, the lower the residual acetic acid concentration is. Thisindicates that both pathways are connected to each other, as was alreadyshown in patent application WO2013/081456.

One of the strains consumed almost all acetic acid (data point lowerleft corner in FIG. 3) and outperformed the other transformants. Thisstrain was designated YD01247.

In total, 2592 strains were screened, including reference strain RN1189.Reference strain RN1189 was included 27 times. The performance ofreference strain RN1189 relative to the other strains (total 2592) isdepicted FIG. 6. The strains are ranked as described before, where thebetter performing strains are indicated by a lighter color (and arecloser to the bottom-left corner of the graph). The less well performingstrains are indicated by a darker color; the change in color is gradual.The exception is that the reference strain, RN1189, is indicated in thedarkest color.

In order to assess the best combinations of expression cassettes,calculations were performed using the NMR data. All strains were scoredon:

-   -   1) the residual acetic acid concentration left in the medium,    -   2) the residual glycerol concentration left in the medium,    -   3) the amount of ethanol produced from glycerol and acetic acid,        by subtracting the theoretical amount of ethanol produced from        xylose and glucose from the total amount of ethanol produced.

All samples were ranked by adding the three scores (table 11). The best150 strains based on this last score was visualized, and is alsodisplayed in FIG. 4.

From the best 150 strains, it was determined which expression cassettes,belonging to the groups A, B, C and D (vide supra), wereoverrepresented. See tables 11 and 12.

TABLE 12 Number of occurrences of each expression cassette in the top150 best performing transformants is depicted in the table below. CAS1 2CAS11 89 CAS13 35 CAS21 38 CAS2 36 CAS12 61 CAS14 37 CAS22 4 CAS3 44CAS15 27 CAS23 51 CAS4 15 CAS16 24 CAS24 39 CAS5 22 CAS17 6 CAS25 9 CAS62 CAS18 9 CAS26 1 CAS7 18 CAS19 11 CAS27 4 CAS8 1 CAS20 1 CAS28 4 CAS9 8CAS10 2

Observations are:

a) in general, the use of strong promoters is counted more frequentlythan the use of weak(er) promoters, as expression cassettes harboringstrong promoters are overrepresented in the 150 strains as compared toweak(er) promoters;

b) CAS2 and CAS3 are overrepresented in the AADH-group;

c) CAS11 and CAS12 are about equally well represented;

d) CAS 13 and CAS 14 seems to be slightly overrepresented in theGLD-group, although the expression cassettes CAS 15 and CAS16 are alsowell represented;

e) CAS 23 is overrepresented in the DAK-group, though CAS21 and CAS23are also well represented in the top 150 strains;

f) all expression cassettes (CAS) that were tested are represented inthe top 150 strains, indicating that multiple solutions exist.

However, many combinations of expression cassettes led to an improvedethanol yield, due to increased glycerol and acetic acid conversion, ascompared to reference strain RN1189. This indicates that many othercombinations of expression cassettes may provide a solution forconverting both glycerol and acetic acid into ethanol, in a mixtureconsisting of these two compounds and fermentable sugars. StrainYD01247, the best strain in the screening (FIGS. 3 and 4) is anillustration of this: it consists of expression cassettes CAS01, CAS12,CAS13 and CAS23.

A number of strains nearly consumed all available acetic acid. Thesestrains were able to consume 3 to 4 grams of glycerol per liter.

Example 3 Retesting of the Best Combinations of Expression Cassettes

In a multifactorial design, the best combinations of expressioncassettes (Example 2) were re-tested. Since in strain YD01247 theexpression of the ACS2-gene was under control of the construct with theweak promoter (i.e. CAS12), this expression cassette was taken along inthe experimental design as well, though it was not overrepresented inthe top 150 strains in Example 2.

Strain RN1069 was transformed with 8 combinations of expressioncassettes selected from groups A, B, C and D (table 13).

TABLE 13 Retesting best combinations of expression cassettesRetransformation Group A Group B Group C Group D R1 CAS02 CAS11 CAS13CAS21 R2 CAS02 CAS11 CAS13 CAS23 R3 CAS02 CAS12 CAS13 CAS21 R4 CAS02CAS12 CAS13 CAS23 R5 CAS03 CAS11 CAS13 CAS21 R6 CAS03 CAS11 CAS13 CAS23R7 CAS03 CAS12 CAS13 CAS21 R8 CAS03 CAS12 CAS13 CAS23

After transformation, cells were spread on YEP-agar supplemented with 20g glucose/liter and 200 μg G418/ml. Per transformation, eightindependent colonies were used to inoculate microplates filled withYEP-agar supplemented with 20 g glucose/liter and 200 μg G418/ml, exceptfor R2; in this case only three transformants were obtained. Asreference strains, RN1069, RN1189 and YD01247 were included, also ineightfold. The strains in the microplate with YEPD-agar and G418 wereused to inoculate 275 μl Mineral Medium containing 200 μg histidine perml, 2% glucose, 2% xylose, 1% glycerol and 2 g/l acetic acid, inmicroplates, in triplicate. The pH of the medium was set at 4.5, belowthe pKa of acetic acid. The microplate was sealed and incubated underanaerobic conditions. At three different time intervals, i.e. after 24,48 and 72 hours, one plate per time point was recovered from theanaerobic shaker. Cells were spun down by centrifugation and thesupernatant was analyzed by NMR. The NMR results, in particular theresidual concentrations of glycerol and acetic acid, after 72 hoursincubation is shown in FIG. 5.

The residual acetic acid concentration in strain RN1069, one of thereference strains, is still close to 2.0 g/l, which is in line with theexpectations. Likewise, this strain also did not consume glycerol.

The proof of concept strain RN1189 (WO2013/081456) consumed in average0.9 gram acetic acid per liter and 1.5 grams of glycerol per liter.

The reference strain YD01247 performed best of all; the residual aceticacid concentration was only 0.2 gram per liter (90% of acetic acidconsumed) and the residual glycerol concentration was only 5.5 gram perliter.

The residual acetic acid concentration of the reconstructedtransformants (except for R2) was between 0.3 and 0.6 gram per liter andthe residual glycerol concentration was between 6.6 and 7.0 grams perliter.

Transformation R2 not only yielded few transformants, but also a largespread was observed in the results. Therefore, these results were notinterpreted.

In conclusion, all combinations of expression cassettes tested, exceptR2, resulted in an improved performance in terms of anaerobic glyceroland acetic acid conversion as compared to the reference strain RN1189.

Example 4 Anaerobic Shake Flask Experiment with Selected Transformants

The performance of a selection of transformants was tested in shakeflasks. To this end, precultures of the strains in table 14, generatedin Example 2, were prepared.

TABLE 14 Strains used in the anaerobic shake flask experiment and thepresence of expression cassettes. Strain GPD1 GPD2 HIS3 AADH ACS GLD DAKCEN.PK113- present present present none none none none 7D RN1069 deleteddeleted deleted none none none none RN1189 deleted deleted comple- adhEEc none gldA Ec DAK1 mented by (plasmid) (plasmid) Sc plasmid (plasmid)YD01247 deleted deleted deleted CAS1 CAS12 CAS13 CAS23 YD01248 deleteddeleted deleted CAS2 CAS11 CAS13 CAS21 YD01249 deleted deleted deletedCAS2 CAS11 CAS13 CAS23 YD01250 deleted deleted deleted CAS2 CAS11 CAS15CAS21 YD01251 deleted deleted deleted CAS2 CAS12 CAS15 CAS23

Strain YD01247, YD01248, YD01249 and YD01250 are indicated in FIG. 7 asnumbers 1, 2, 3 and 4 respectively. Strain YD01251 is a strain thatperforms medium well in terms of glycerol and acetic acid consumption.

100 ml shake flask were filled with 25 ml of Mineral Medium (asdescribed in Material and Methods) containing approximately per liter:20 grams of glucose, 20 grams of xylose, 10 grams of glycerol, 200 mghistidine and 2 grams of acetic acid (HAc), pH 4.5.

These shake flasks were inoculated, in duplicate, with washed cells fromthe precultures with the amount needed to achieve an initial OD600 of0.5. The flasks were closed with waterlocks in order to achieveanaerobic conditions during the fermentation. The incubation was done at32° C. and 100 rpm. After 96 hours, the fermentation was terminated andcells were spun down by centrifugation. The supernatant was analyzed byNMR. The results are shown in table 15.

TABLE 15 Averaged NMR results of the shake anaerobic flask experimentEthanol yield on Acetic consumed Glucose Xylose Glycerol acid Ethanolsugars (g/l) (g/l) (g/l) (g/l) (g/l) (g/g) Medium 21.1 19.9 9.2 2.0 0.20.00 CEN.PK113- 0.6 20.3 9.2 2.0 9.1 0.44 7D RN1069 2.3 12.9 9.3 1.710.1 0.39 RN1189 0.1 2.0 7.4 1.4 18.8 0.48 YD01247 0.1 6.5 4.8 0.2 17.10.50 YD01248 0.2 1.4 5.0 0.3 19.5 0.49 YD01249 0.2 1.2 5.3 0.4 19.4 0.49YD01250 0.1 0.8 5.4 0.4 19.4 0.48 YD01251 0.0 1.6 5.8 0.6 19.0 0.48

Strain CEN.PK113-7D only ferments glucose, which is in line with theexpectations. The ethanol yield, calculated on basis of consumed sugars,is 0.44, which is in concordance with what is usually found in shakeflask fermentations. The theoretical maximum ethanol yield amounts 0.51grams of ethanol per gram of sugar.

Strain RN1069 can, in principle, ferment both glucose and xylose.However, this strain has a deletion of both GPD1 and GPD2, whichdisables co-factor regeneration under anaerobic conditions. Yet, thisstrain converts glucose and xylose partly, presumably because at thestart of the experiment, some residual oxygen was available in the headspace of the shake flask, as well as dissolved oxygen in the medium,enabling some cofactor recycling in the beginning of the experiment.Yet, the ethanol yield is low, 0.39 grams of ethanol per gram ofconsumed sugar.

The transformed strains expressing AADH, GLD and DAK from a plasmid(RN1189) or expressing AADH, ACS, GLD and DAK from an integratedconstruct in the genome (YD01247 through YD01251) show an increasedethanol yield per consumed sugar. Ethanol yields of 0.48 up to 0.50 areachieved. These higher values were achieved due to the anaerobicconversion of glycerol and acetic acid into ethanol and/or the absenceof glycerol production.

Strain YD01247 has consumed less xylose than the other YD-strains.However, this strain has consumed almost all acetic acid, as was alreadyshown in Examples 2 and 3. This strain has also consumed most of theglycerol. This strain showed the highest ethanol yield.

The other YD-strains also show an improved performance relative tostrain RN1189: more glycerol and acetic acid were consumed, leading to ahigher ethanol titer.

These experiments show that various alternative combinations ofexpression cassettes resulted in an improved performance in terms ofanaerobic glycerol and acetic acid conversion as compared to thereference strain RN1189, resulting in a higher ethanol titer in allcases and a higher ethanol yield in some cases.

LITERATURE

-   Engler C. et al (2011) Generation of families of construct variants    using golden gate shuffling. Methods Mol Biol. 2011; 729:1, 67-81.    doi: 10.1007/978-1-61779-065-2_11;-   Guadalupe Medina V, Almering M J, van Maris A J, Pronk J T (2009)    “Elimination of glycerol production in anaerobic cultures of    Saccharomyces cerevisiae engineered for use of acetic acid as    electron acceptor.” Appl Environ Microbiol.;-   Lee and Dasilva (2006) Application of sequential integration for    metabolic engineering of 1,2-propanediol production in yeast. Metab    Eng. 8(1):58-65;-   Sonderegger et al (2004) Metabolic Engineering of a Phosphoketolase    Pathway for Pentose Catabolism in Saccharomyces cerevisiae. AEM    70(5), 2892-2897;-   Van Dijken and Scheffers (1986) Redox balances in the metabolism of    sugars by yeasts. FEMS Microbiology Letters Volume 32, Issue 3-4,    pages 199-224;-   Yu et al (2010) Engineering of glycerol utilization pathway for    ethanol production by Saccharomyces cerevisiae. Bioresour. Technol.    101(11):4157-4161;-   Yu et al (2011) Improvement of ethanol yield from glycerol via    conversion of pyruvate to ethanol in metabolically engineered    Saccharomyces cerevisiae. Appl Biochem Biotecnol    doi:10.1007/s12010-011-9475-9;-   Patent application PCT/EP2013/056623 (non-published);-   Patent application WO 2013/081456;-   Patent application WO 2011/010923.

1. A cell that is genetically modified comprising: a) one or morenucleotide sequence encoding a NAD₊-dependent acetylating acetaldehydedehydrogenase (E.C. 1.2.1.10); b) one or more nucleotide sequenceencoding a acetyl-CoA synthetase (E.C. 6.2.1.1); c) one or morenucleotide sequence encoding a glycerol dehydrogenase (E.C. 1.1.1.6);and d) one or more nucleotide sequence encoding a dihydroxyacetonekinase (E.C. 2.7.1.28 or E.C. 2.7.1.29).
 2. The cell according to claim1, comprising a deletion or disruption of one or more endogenousnucleotide sequence encoding a glycerol 3-phosphate phosphohydrolaseand/or encoding a glycerol 3-phosphate dehydrogenase gene.
 3. The cellaccording to claim 2, wherein c) is one or more nucleotide sequenceencoding a heterologous glycerol dehydrogenase (E.C. 1.1.1.6)represented by SEQ ID NO: 7 or a functional homologue of SEQ ID NO: 7having sequence identity of at least 60% with SEQ ID NO: 7; and/or oneor more nucleotide sequence encoding a heterologous glyceroldehydrogenase (E.C. 1.1.1.6) represented by SEQ ID NO: 9 or a functionalhomologue of SEQ ID NO: 9 having sequence identity of at least 60% withSEQ ID NO:
 9. 4. The cell according to claim 3, wherein c) is one ormore nucleotide sequence encoding a heterologous glycerol dehydrogenase(E.C. 1.1.1.6) represented by SEQ ID NO: 7 or a functional homologue ofSEQ ID NO: 7 having sequence identity of at least 60% with SEQ ID NO: 7.5. The cell according to claim 1, wherein d) is one or more heterologousnucleotide sequence encoding a dihydroxyacetone kinase (E.C. 2.7.1.28 orE.C. 2.7.1.29) represented by SEQ ID NO: 11 or a functional homologue ofSEQ ID NO: 11 having sequence identity of at least 40% with SEQ ID NO:11 and/or one or more nucleotide sequence encoding a dihydroxyacetonekinase (E.C. 2.7.1.28 or E.C. 2.7.1.29) represented by SEQ ID NO: 13 ora functional homologue of SEQ ID NO: 13 having sequence identity of atleast 40% with SEQ ID NO:
 13. 6. The cell according to claim 5, whereind) is one or more nucleotide sequence encoding a dihydroxyacetone kinase(E.C. 2.7.1.28 or E.C. 2.7.1.29) represented by SEQ ID NO: 13 or afunctional homologue of SEQ ID NO: 13 having sequence identity of atleast 40% with SEQ ID NO:
 13. 7. The cell according to claim 1 whereina) is one or more nucleotide sequence encoding a heterologousNAD₊-dependent acetylating acetaldehyde dehydrogenase represented by SEQID NO: 1 or a functional homologue of SEQ ID NO: 1 having sequenceidentity of at least 60% with SEQ ID NO: 1; and/or one or morenucleotide sequence encoding a heterologous NAD₊-dependent acetylatingacetaldehyde dehydrogenase represented by SEQ ID NO: 2 or a functionalhomologue of SEQ ID NO: 2 having sequence identity of at least 60% withSEQ ID NO: 2 and/or one or more nucleotide sequence encoding aheterologous NAD₊-dependent acetylating acetaldehyde dehydrogenaserepresented by SEQ ID NO: 3 or a functional homologue of SEQ ID NO: 3having sequence identity of at least 60% with SEQ ID NO:
 3. 8. The cellaccording to claim 7, wherein a) is one or more nucleotide sequenceencoding a heterologous NAD₊-dependent acetylating acetaldehydedehydrogenase represented by SEQ ID NO: 1 or a functional homologue ofSEQ ID NO: 1 having sequence identity of at least 60% with SEQ ID NO: 1;and/or one or more nucleotide sequence encoding a heterologousNAD₊-dependent acetylating acetaldehyde dehydrogenase represented by SEQID NO: 2 or a functional homologue of SEQ ID NO: 2 having sequenceidentity of at least 60% with SEQ ID NO:
 2. 9. The cell according toclaim 8 wherein a) is one or more nucleotide sequence encoding aheterologous NAD₊-dependent acetylating acetaldehyde dehydrogenaserepresented by SEQ ID NO: 2 or a functional homologue of SEQ ID NO: 2having sequence identity of at least 60% with SEQ ID NO:
 2. 10. The cellaccording to claim 1, wherein b) is one or more nucleotide sequenceencoding a homologous or heterologous acetyl-CoA synthetase (E.C.6.2.1.1) represented by SEQ ID NO: 6 or a functional homologue of SEQ IDNO: 6 having sequence identity of at least 60% with SEQ ID NO:
 6. 11.The cell according to claim 1 wherein a) is one or more nucleotidesequence encoding a NAD₊-dependent acetylating acetaldehydedehydrogenase represented by SEQ ID NO: 3 or a functional homologue ofSEQ ID NO: 3 having sequence identity of at least 60% with SEQ ID NO: 3;b) is one or more nucleotide sequence encoding a homologous orheterologous acetyl-CoA synthetase (E.C. 6.2.1.1) represented by SEQ IDNO: 6 or a functional homologue of SEQ ID NO: 6 having sequence identityof at least 60% with SEQ ID NO: 6; c) is one or more nucleotide sequenceencoding a glycerol dehydrogenase (E.C. 1.1.1.6) represented by SEQ IDNO: 7 or a functional homologue of SEQ ID NO: 7 having sequence identityof at least 60% with SEQ ID NO: 7; and d) is one or more nucleotidesequence encoding a homologous or heterologous dihydroxyacetone kinase(E.C. 2.7.1.28 or E.C. 2.7.1.29) represented by SEQ ID NO: 11 or afunctional homologue of SEQ ID NO: 11 having sequence identity of atleast 40% with SEQ ID NO:
 11. 12. The cell according to claim 1, whereina) is one or more nucleotide sequence encoding a NAD₊-dependentacetylating acetaldehyde dehydrogenase represented by SEQ ID NO: 2 or afunctional homologue of SEQ ID NO: 2 having sequence identity of atleast 60% with SEQ ID NO: 2; b) is one or more nucleotide sequenceencoding a homologous or heterologous acetyl-CoA synthetase (E.C.6.2.1.1) represented by SEQ ID NO: 6 or a functional homologue of SEQ IDNO: 6 having sequence identity of at least 60% with SEQ ID NO: 6; c) isone or more nucleotide sequence encoding a glycerol dehydrogenase (E.C.1.1.1.6) represented by SEQ ID NO: 9 or a functional homologue of SEQ IDNO: 9 having sequence identity of at least 60% with SEQ ID NO: 9; and d)is one or more nucleotide sequence encoding a homologous or heterologousdihydroxyacetone kinase (E.C. 2.7.1.28 or E.C. 2.7.1.29) represented bySEQ ID NO: 11 or a functional homologue of SEQ ID NO: 11 having sequenceidentity of at least 40% with SEQ ID NO:
 11. 13. The cell according toclaim 1, wherein a) is one or more heterologous nucleotide sequenceencoding a NAD₊-dependent acetylating acetaldehyde dehydrogenaserepresented by SEQ ID NO: 2 or a functional homologue of SEQ ID NO: 2having sequence identity of at least 60% with SEQ ID NO: 2; b) is one ormore nucleotide sequence encoding a homologous or heterologousacetyl-CoA synthetase (E.C. 6.2.1.1) represented by SEQ ID NO: 6 or afunctional homologue of SEQ ID NO: 6 having sequence identity of atleast 60% with SEQ ID NO: 6; c) is one or more nucleotide sequenceencoding a glycerol dehydrogenase (E.C. 1.1.1.6) represented by SEQ IDNO: 7 or a functional homologue of SEQ ID NO: 7 having sequence identityof at least 60% with SEQ ID NO: 7; and d) is one or more nucleotidesequence encoding a homologous or heterologous dihydroxyacetone kinase(E.C. 2.7.1.28 or E.C. 2.7.1.29) represented by SEQ ID NO: 13 or afunctional homologue of SEQ ID NO: 13 having sequence identity of atleast 40% with SEQ ID NO:
 13. 14. The cell according to claim 1, whereina) is one or more nucleotide sequence encoding a NAD₊-dependentacetylating acetaldehyde dehydrogenase represented by SEQ ID NO: 1 or afunctional homologue of SEQ ID NO: 1 having sequence identity of atleast 60% with SEQ ID NO: 1; b) is one or more nucleotide sequenceencoding a acetyl-CoA synthetase (E.C. 6.2.1.1) represented by SEQ IDNO: 6 or a functional h homologous or heterologous omologue of SEQ IDNO: 6 having sequence identity of at least 60% with SEQ ID NO: 6; c) isone or more heterologous nucleotide sequence encoding a glyceroldehydrogenase (E.C. 1.1.1.6) represented by SEQ ID NO: 7 or a functionalhomologue of SEQ ID NO: 7 having sequence identity of at least 60% withSEQ ID NO: 7; and d) is one or more nucleotide sequence encoding a dihomologous or heterologous hydroxyacetone kinase (E.C. 2.7.1.28 or E.C.2.7.1.29) represented by SEQ ID NO: 13 or a functional homologue of SEQID NO: 13 having sequence identity of at least 40% with SEQ ID NO: 13.15. The cell according to claim 1, wherein the cell is a yeast cell. 16.The cell according to claim 15, wherein in the yeast cell all endogenousnucleotide sequences encoding a glycerol 3-phosphate phosphohydrolaseand all endogenous nucleotide sequences encoding a glycerol 3-phosphatedehydrogenase are deleted.
 17. The cell according to claim 16, whereinthe yeast cell is free of genes encoding NADH-dependent glycerol3-phosphate dehydrogenase.
 18. The yeast cell according to claim 15,wherein the yeast cell is selected from the group consisting ofSaccharomycetaceae, optionally from the group of Saccharomyces,optionally Saccharomyces cerevisiae; Kluyveromyces, optionallyKluyveromyces marxianus; Pichia, optionally Pichia stipitis or Pichiaangusta; Zygosaccharomyces, optionally Zygosaccharomyces bailii; andBrettanomyces, optionally Brettanomyces intermedius, Issatchenkia,optionally Issatchenkia orientalis and Hansenula.
 19. The cell accordingto claim 1, wherein the cell is a prokaryotic cell.
 20. The cellaccording to claim 19, wherein the cell is selected from the groupconsisting of Clostridium, Zymomonas, Thermobacter, Escherichia,Lactobacillus, Geobacillus and Bacillus.
 21. A polynucleotide encoding apolypeptide represented by any of SEQ ID NO: 1 to14.
 22. A nucleic acidconstruct comprising one or more polynucleotide of claim
 21. 23. A hostcell transformed with the nucleic acid construct of claim
 22. 24. A cellaccording to claim 1 capable of being used for the preparation ofethanol.
 25. A process for preparing fermentation product, comprisingpreparing a fermentation product from acetate and from a fermentablecarbohydrate—optionally a carbohydrate selected from the group ofglucose, fructose, sucrose, maltose, xylose, arabinose, galactose andmannose which preparation is carried out under anaerobic conditionsusing a yeast cell, according to claim
 18. 26. The process according toclaim 25, wherein the preparation is carried out in a fermentationmedium comprising the acetate and the carbohydrate in a molar ratio is0.7 or less, optionally at least 0.004 to 0.5, optionally 0.05 to 0.3.27. The process according to claim 25, wherein at least part of thecarbohydrate and at least part of the acetate has been obtained byhydrolysing a polysaccharide selected from the group of lignocelluloses,celluloses, hemicelluloses, and pectins.
 28. The process according toclaim 27, wherein the lignocelluloses is lignocellulosic biomass thathas been hydrolysed thereby obtaining the fermentable carbohydrate andacetate.
 29. The process according to claim 28, wherein lignocellulosicor hemi-cellulosic material is contacted with an enzyme composition,wherein one or more sugar is produced, and wherein the produced sugar isfermented to give a fermentation product, wherein the fermentation isconducted with a transformed host cell of any of claims 1 to
 18. 30. Theprocess according to claim 29, wherein the fermentation product is oneor more of ethanol, butanol, lactic acid, a plastic, an organic acid, asolvent, an animal feed supplement, a pharmaceutical, a vitamin, anamino acid, an enzyme or a chemical feedstock.